CA2457043A1 - Medicinal use of histone deacetylase inhibitor and method of evaluating antitumor effect thereof - Google Patents
Medicinal use of histone deacetylase inhibitor and method of evaluating antitumor effect thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
Remedies for prostatic cancer and malignant lymphoma comprising FK228 or its salt as the active ingredient; and a method of evaluating the antitumor effect of a histone deacetylase inhibitor characterized by comprising at least the step of treating test cells with the histone deacetylase inhibitor and the step of measuring a change in the expression of a specific gene in the test cells before and after the treatment with the inhibitor and comparing the expression doses thus measured.
Description
DESCRIPTION
MEDICINAL USE OF HISTONE DEACETYLASE INHIBITOR AND METHOD OF
EVALUATING ANTITUMOR EFFECT THEREOF
Technical Field The present invention relates to a therapeutic agent for prostate cancer, a therapeutic agent for malignant lymphoma (except T cell lymphoma), and a method of evaluating an antitumor effect of histone deacetylase inhibitors.
Background Art to In recent years, "tailor made medicine" is gaining recognition, which takes into consideration individual differences between patients, and a search for a marker to distinguish a cancer against which a pharmaceutical agent is effective from a cancer against which the pharmaceutical agent 15 is ineffective is considered to be necessary. It is an attempt to ethically and medically improve cost performance of medication treatment by administering a pharmaceutical agent to patients after verification in advance of the probability of effect thereof, thereby to enhance efficacy as well as 2o avoid toxicity of the pharmaceutical agent, and to reduce insignificant use of the pharmaceutical agent. In cancer treatment, the development of a method for predicting the efficacy of anticancer agents has been desired, because it can be an important means to bridge the gap between basic study 2s and clinical application.
In addition, it has been pointed out with regard to a substance or a compound generally reported to have an antitumor activity that, when the report is based solely on in vitro results, such results do not directly lead to the 3o prediction of in vivo results. In other words, it is a problem that a substance showing an antitumor activity in vitro does not necessarily show an antitumor activity in vivo, and application of a substance showing an antitumor activity in DOCSTOR: 639063\1 vitro directly as an anticancer agent is difficult.
For example, a compound represented by the formula (II) O
HN
NH
O S
HN S O
(II) O NH
O
has been reported to introduce a potent antitumor activity by .s selectively inhibiting histone deacetylase (this substance has been also reported to cause high acetylation of histone in a cell treated with this substance, and as a result, induces transcriptional control activity of various genes, cell cycle inhibitory activity and apoptosis inhibitory activity (JP-B-7-l0 64872, H. Nakajima et al, Exp. Cell Res. 241, 126-133 (1998))).
However, no report has established a factor capable of predicting an antitumor effect of this compound, and as the situation stands, many problems are yet to be solved, such as whether or not in vitro results directly apply in vivo, Is whether or not the compound shows a practical effect in vivo in any tumor and the like.
Histone deacetylase is a metallo-deacetylated enzyme wherein Zn is coordinated at the active center (M.S. Finnin et al, Nature, 401, 188-193 (1999)). This enzyme is considered to zo change the affinity for DNA of various acetylated histones. A
direct biological phenomenon this brings about is changes in the chromatin structure. The minimum unit of the chromatin structure is a nucleosome wherein 146 by DNA is wound 1.8 DOCSTOR: 639063\1 times anticlockwise around a histone octamer (H2A, H2B, H3 and H4, each 2 molecules, core histone?. The core histone stabilizes the nucleosome structure as the positive charge at the N-terminal of each histone protein interacts with DNA.
s Acetylation of histone is controlled by the balance between the acetylation reaction in which histone acetyl transferase is involved and the deacetylation reaction in which histone deacetylase is involved. The acetylation of histone occurs in an evolutionarily well-preserved lysine residue at the N-to terminal of histone protein, whereby, it is considered, the core histone protein loses the charge at the N-terminal, the interaction with DNA is attenuated, and the nucleosome structure becomes instable. Accordingly, the deacetylation of histone is considered to proceed in reverse, namely, toward 15 the stabilization of the nucleosome structure. However, there still remain many unclear aspects such as the degree the acetylation changes the chromatin structure, and how it relates to the secondarily induced transcriptional control and the like.
2o Disclosure of the Invention An object of the present invention is to provide a novel therapeutic agent for prostate cancer and a therapeutic agent for malignant lymphoma. Another object of the present invention is to provide a method for evaluating and predicting z5 an antitumor effect of a histone deacetylase inhibitor.
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems and found a therapeutic agent for prostate cancer and a therapeutic agent for malignant lymphoma that permit confirmation of in vivo 3o antitumor effect. Moreover, the present inventors have found that an antitumor effect of a histone deacetylase inhibitor varies depending on the kind of tumor, and that the variation is observed in conjunction with changes in the expression DOCSTOR: 639063'1 state of a specific gene or protein, and based on such observation, established a method for evaluating an antitumor effect of a histone deacetylase inhibitor, which resulted in the completion of the present invention. Accordingly, the s present invention provides the following.
(1) An agent for treating prostate cancer or an agent for treating malignant lymphoma other than T cell lymphoma, which comprises, as an active ingredient, a compound represented by the formula (I) (hereinafter to be also referred to as FK228;
1o SEQ ID; No 5) / \H~C-.... N\ l/CH
O~. ~N C~ C \ /O
C S C
CH3 I (I) CH - CH S NH
CH3 1 ~CHz HN~ HZC CH w CH
CH ~ I ~ CH3 C ~i C
HC ~ \ CH3 O H~C~O O
, particularly a compound represented by the formula (II) (hereinafter to be also referred to as FR901228) O
HN
NH
O S
HN ~ O
(II) O NH
J
MEDICINAL USE OF HISTONE DEACETYLASE INHIBITOR AND METHOD OF
EVALUATING ANTITUMOR EFFECT THEREOF
Technical Field The present invention relates to a therapeutic agent for prostate cancer, a therapeutic agent for malignant lymphoma (except T cell lymphoma), and a method of evaluating an antitumor effect of histone deacetylase inhibitors.
Background Art to In recent years, "tailor made medicine" is gaining recognition, which takes into consideration individual differences between patients, and a search for a marker to distinguish a cancer against which a pharmaceutical agent is effective from a cancer against which the pharmaceutical agent 15 is ineffective is considered to be necessary. It is an attempt to ethically and medically improve cost performance of medication treatment by administering a pharmaceutical agent to patients after verification in advance of the probability of effect thereof, thereby to enhance efficacy as well as 2o avoid toxicity of the pharmaceutical agent, and to reduce insignificant use of the pharmaceutical agent. In cancer treatment, the development of a method for predicting the efficacy of anticancer agents has been desired, because it can be an important means to bridge the gap between basic study 2s and clinical application.
In addition, it has been pointed out with regard to a substance or a compound generally reported to have an antitumor activity that, when the report is based solely on in vitro results, such results do not directly lead to the 3o prediction of in vivo results. In other words, it is a problem that a substance showing an antitumor activity in vitro does not necessarily show an antitumor activity in vivo, and application of a substance showing an antitumor activity in DOCSTOR: 639063\1 vitro directly as an anticancer agent is difficult.
For example, a compound represented by the formula (II) O
HN
NH
O S
HN S O
(II) O NH
O
has been reported to introduce a potent antitumor activity by .s selectively inhibiting histone deacetylase (this substance has been also reported to cause high acetylation of histone in a cell treated with this substance, and as a result, induces transcriptional control activity of various genes, cell cycle inhibitory activity and apoptosis inhibitory activity (JP-B-7-l0 64872, H. Nakajima et al, Exp. Cell Res. 241, 126-133 (1998))).
However, no report has established a factor capable of predicting an antitumor effect of this compound, and as the situation stands, many problems are yet to be solved, such as whether or not in vitro results directly apply in vivo, Is whether or not the compound shows a practical effect in vivo in any tumor and the like.
Histone deacetylase is a metallo-deacetylated enzyme wherein Zn is coordinated at the active center (M.S. Finnin et al, Nature, 401, 188-193 (1999)). This enzyme is considered to zo change the affinity for DNA of various acetylated histones. A
direct biological phenomenon this brings about is changes in the chromatin structure. The minimum unit of the chromatin structure is a nucleosome wherein 146 by DNA is wound 1.8 DOCSTOR: 639063\1 times anticlockwise around a histone octamer (H2A, H2B, H3 and H4, each 2 molecules, core histone?. The core histone stabilizes the nucleosome structure as the positive charge at the N-terminal of each histone protein interacts with DNA.
s Acetylation of histone is controlled by the balance between the acetylation reaction in which histone acetyl transferase is involved and the deacetylation reaction in which histone deacetylase is involved. The acetylation of histone occurs in an evolutionarily well-preserved lysine residue at the N-to terminal of histone protein, whereby, it is considered, the core histone protein loses the charge at the N-terminal, the interaction with DNA is attenuated, and the nucleosome structure becomes instable. Accordingly, the deacetylation of histone is considered to proceed in reverse, namely, toward 15 the stabilization of the nucleosome structure. However, there still remain many unclear aspects such as the degree the acetylation changes the chromatin structure, and how it relates to the secondarily induced transcriptional control and the like.
2o Disclosure of the Invention An object of the present invention is to provide a novel therapeutic agent for prostate cancer and a therapeutic agent for malignant lymphoma. Another object of the present invention is to provide a method for evaluating and predicting z5 an antitumor effect of a histone deacetylase inhibitor.
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems and found a therapeutic agent for prostate cancer and a therapeutic agent for malignant lymphoma that permit confirmation of in vivo 3o antitumor effect. Moreover, the present inventors have found that an antitumor effect of a histone deacetylase inhibitor varies depending on the kind of tumor, and that the variation is observed in conjunction with changes in the expression DOCSTOR: 639063'1 state of a specific gene or protein, and based on such observation, established a method for evaluating an antitumor effect of a histone deacetylase inhibitor, which resulted in the completion of the present invention. Accordingly, the s present invention provides the following.
(1) An agent for treating prostate cancer or an agent for treating malignant lymphoma other than T cell lymphoma, which comprises, as an active ingredient, a compound represented by the formula (I) (hereinafter to be also referred to as FK228;
1o SEQ ID; No 5) / \H~C-.... N\ l/CH
O~. ~N C~ C \ /O
C S C
CH3 I (I) CH - CH S NH
CH3 1 ~CHz HN~ HZC CH w CH
CH ~ I ~ CH3 C ~i C
HC ~ \ CH3 O H~C~O O
, particularly a compound represented by the formula (II) (hereinafter to be also referred to as FR901228) O
HN
NH
O S
HN ~ O
(II) O NH
J
DOCSTOR: 639063\1 or a salt thereof.
(2) The agent for treating prostate cancer or the agent for treating malignant lymphoma other than T cell lymphoma of the above-mentioned (1), which has an antitumor action in Vivo.
s (3) A pharmaceutical composition for treating prostate cancer or a pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma, which comprises FK228, particularly the formula FR901228, and a pharmaceutically acceptable carrier.
to (4) The pharmaceutical composition for treating prostate cancer or the pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma of the above-mentioned (3), which has an antitumor action in vivo.
(2) The agent for treating prostate cancer or the agent for treating malignant lymphoma other than T cell lymphoma of the above-mentioned (1), which has an antitumor action in Vivo.
s (3) A pharmaceutical composition for treating prostate cancer or a pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma, which comprises FK228, particularly the formula FR901228, and a pharmaceutically acceptable carrier.
to (4) The pharmaceutical composition for treating prostate cancer or the pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma of the above-mentioned (3), which has an antitumor action in vivo.
(5) A method for treating prostate cancer or malignant Is lymphoma other than T cell lymphoma, which comprises administering an effective amount of FK228, particularly the formula FR901228.
(6) Use of FK228, particularly the formula FR901228, for the production of an agent for treating prostate cancer or an 2o agent for treating malignant lymphoma other than T cell lymphoma.
(7) The use of the above-mentioned (6), wherein the agent for treating prostate cancer or the agent for treating malignant lymphoma other than T cell lymphoma has an antitumor action in 2s V1 V0.
(8) A commercial package comprising the pharmaceutical composition for treating prostate cancer of the above-mentioned (3) and a written matter stating that the pharmaceutical composition can or should be used for treating so prostate cancer.
(9) A commercial package comprising the pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma of the above-mentioned (3) and a written matter DOCSTOR: 639063U
stating that the pharmaceutical composition can or should be used for treating malignant lymphoma other than T cell lymphoma.
stating that the pharmaceutical composition can or should be used for treating malignant lymphoma other than T cell lymphoma.
(10) A method for evaluating an antitumor effect of a histone s deacetylase inhibitor, which comprises at least a step of treating a test cell with a histone deacetylase inhibitor, and a step of measuring change in the expression of a specific gene (or specific protein) in the test cell before and after the treatment with said inhibitor, and comparing the both so expression amounts.
(11) The method for evaluating an antitumor effect of a histone deacetylase inhibitor of the above-mentioned (10), wherein the specific gene is a p21 gene and/or a c-myc gene.
(12) The method for evaluating an antitumor effect of a 15 histone deacetylase inhibitor of the above-mentioned (10), wherein the specific protein i~s a p21 gene and/or a c-myc gene.
(13) The method for evaluating an antitumor effect of any of the above-mentioned (10)-(12), wherein the histone deacetylase inhibitor is a compound represented by the formula FK228, 2o particularly FR901228, or a salt thereof.
(14) A method for screening a histone deacetylase inhibitor having a site-specific antitumor activity, which comprises use of the method for evaluating an antitumor effect of any of the above-mentioned (10)-(13).
2s (15) A method for obtaining a gene capable of becoming an index for predicting the efficacy of FK228, which comprises at least (1) a step of treating an FK228 sensitive tumor cell and an FK228 resistant tumor cell with FK228, 30 (2) a step of selecting genes that show increased or decreased expression in step (1) above, and (3) a step of selecting, from the genes selected in step (2) above, DOCSTOR: 639063\ 1 (i) a gene that shows increased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, s (ii) a gene that shows increased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell, (iii) a gene that shows decreased expression due to the to treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, or (iv) a gene that shows decreased expression due to the treatment with FK228, lower expression in the FK228 Is sensitive tumor cell and higher expression in the FK228 resistant tumor cell.
Brief Description of the Drav~rings Fig. 1 is a graph showing an antitumor effect of FR901228 on human prostate cancer, wherein the vertical axis 2o shows a tumor growth rate, the transverse axis shows the number of days lapsed from the initial administration, and the tumor growth rate is expressed in a relative proportion of tumor volume after day 0 relative to the tumor volume on day 0 taken as 1.
2s Fig. 2 includes graphs showing an antitumor effect of FR901228 on human lymphoma, wherein the vertical axis shows the proportion of survived mice, and the transverse axis shows the number of days lapsed after tumor cell implantation.
so Fig. 3 includes graphs showing an antitumor effect of FR901228 on human prostate cancer ((a); PC-3) and kidney cancer ((b): ACHN), wherein the vertical axis shows a tumor growth rate, the transverse axis shows the number of days DOCSTOR: 639063\1 lapsed after the initial administration, and the tumor growth rate is expressed in a relative proportion of tumor volume after day 0 relative to the tumor volume on day 0 taken as 1.
s Fig. 4 includes graphs showing an action of FR901228 on p21 gene expression in vitro (PC-3 cell, ACHN cell).
(a),(b); The vertical axis shows a relative amount of p21 gene expression, and the transverse axis shows contact time (hr) with FR901228.
to (c); The vertical axis shows a relative amount of p21 gene expression.
Fig. 5 includes graphs showing an action of FR901228 on p21 gene expression and c-myc gene expression in vivo (PC-3 cell, ACHN cell), wherein the vertical axis shows a 15 relative amount of p21 or c-myc gene expression, and the transverse axis shows the number of days lapsed after administration of FR901228.
Detailed Description of the Invention The therapeutic agent for prostate cancer and the 2o therapeutic agent for malignant lymphoma of the present invention comprise, as an active ingredient, a compound (FK228) represented by the formula (I) or a salt thereof. Of the compounds of the formula (I), a compound (FR901228) represented by the formula (II), which is a stereoisomer, is 25 preferable. These compounds have a strong histone deacetylase inhibitory activity (Nakajima, H. et al; ibid.
(1998)), and FR901228 is particularly preferably contained in the therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present 3o invention, because it has a stronger histone deacetylase inhibitory activity.
In the present specification, a simple reference to FK228 means a group of compounds regardless of stereoisomerism, DOCSTOR: 639063\1 including the compounds represented by the formula (II), unless otherwise specified.
FK228 and a salt thereof are known and available substances. For example, FR901228, which is one of the s stereoisomers of FK228, can be obtained by culturing a strain capable of producing FR901228 and belonging to the genus Chromobacterium under aerobic conditions and recovering the substance from the culture broth. As the strain capable of producing FR901228 and belonging to the genus Chromobacterium, io for example, Chromobacterium violaceum WB968 (FERM BP-1968) can be mentioned. More specifically, FR901228 can be obtained from a FR901228-producing strain according to the method described in JP-B-7-64872 (corresponding to US Patent No.
4977138). FR901228 is preferably recovered from a strain i5 capable of producing FR901228 and belonging to the genus Chromobacterium, because it is obtained more easily. However, synthetic or semi-synthetic FR901228 is also advantageous, because no or only fewer steps of purification is/are required.
Likewise, FK228 other than FR901228 can be also semi-ao synthesized or totally synthesized according to a conventionally known method. More specifically, it can be produced according to the method reported by Khan W.Li, et al (J. Am. Chem. Soc., vol. 118, 7237-7238 (1996)).
The salt of FK228 is a biologically acceptable, generally 2s nontoxic salt, and examples thereof include salts with inorganic base (e. g., alkali metal salts such as sodium salt, potassium salt etc., alkaline earth metal salts such as calcium salt, magnesium salt etc., and ammonium salt), salts with organic base (e.g., organic amine salts such as 3o triethylamine salt, diisopropylethyl amine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt etc.), inorganic acid addition salts (e. g., hydrochloride, DOCSTOR: 639063\1 hydrobromide, hydrosulfate, phosphate etc.), organic carboxylic acid ~ sulfonic acid addition salts (e. g., formate, acetate, trifluoroacetate, maleate, tartrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate etc.), s salts with a basic or acidic amino acid (e. g., arginine, aspartic acid, glutamic acid etc.), salts with a base and acid addition salts.
FK228 may have a stereoisomer (e.g., FR901228) such as an optical isomer or geometric isomer based on an asymmetric ~o carbon atom or a double bond, and all the isomers and a mixture thereof are within the scope of the present invention.
Solvate compounds of FK228, FR901228 and salts thereof (e. g., inclusion compounds (e. g., hydrate etc.)) are also encompassed in the scope of the present invention.
Is In the present invention, in vivo and in vitro generally mean as used in the pertinent field. That is, "in vivo" refers to a state where an object biological function or reaction is expressed in the living organism, and "in vitro" refers to an expression of such function or reaction in a test tube (tissue 2o culture system, cell culture system, cell free system etc.).
The tumor to be the target in the present invention is a tumor on which FK228, which is a histone deacetylase inhibitor, exerts an antitumor effect, and examples thereof include prostate cancer and malignant lymphoma, where in vivo effect 2s is particularly remarkable. The malignant lymphoma, on which the therapeutic agent for malignant lymphoma of the present invention shows an antitumor effect, is preferably that other than T cell lymphoma, such as B cell lymphoma, histiocytosis lymphoma and the like. The present invention shows fine 3o antitumor effect in vivo particularly against these tumors.
The therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present invention can be used as a pharmaceutical preparation in the DOCSTOR: 639063\1 form of a solid, semi-solid or liquid containing FK228 or a salt thereof as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for oral or parenteral application. The active ingredient can be admixed s with a conventional, nontoxic, pharmaceutically acceptable carrier for, for example, powder, tablet, pellet, capsule, suppository, liquid, emulsion, suspension, aerosol, spray and other form suitable for use. Where necessary, auxiliary, stabilizer, thickening agent and the like may be also used.
so These carriers and excipients may be used after a sterilization treatment where necessary, or may be sterilized after formulation into a preparation. FK228 or a salt thereof may be contained in an amount sufficient to provide an antitumor effect, in the therapeutic agent for prostate cancer Is and the therapeutic agent for malignant lymphoma.
When the pharmaceutical agent is applied to a human, it is preferably applied by intravenous, intramuscular or oral administration. While the therapeutically effective dose of FK228 or a salt thereof, which are active ingredients, varies 2o depending on the age and condition of individual patients to be treated, and the kind of cancer and the kind of malignant lymphoma, it is generally 0.1-100 mg, preferably 1-50 mg, more preferably 5-30 mg, a day in the amount of FK228 per human body surface area (m2) in the case of intravenous 2s administration for the treatment of tumor.
The present invention also provides an evaluation method of an antitumor effect of the histone deacetylase inhibitor.
Using this method, a histone deacetylase inhibitor that can exert an antitumor effect on the target tumor cell can be 3o found without actually administering the inhibitor to a human body.
By the "histone deacetylase inhibitor" is meant a compound that binds to the active site of histone deacetylase DOCSTOR: 639063\1 competitively with substrate, or a compound that binds to a site other than the active site of histone deacetylase and has an action to alter the enzyme activity of histone deacetylase, and it encompasses a compound already known as a histone s deacetylase inhibitor whose use is known, all compounds (synthetic or natural) reported to have a histone deacetylase inhibitory activity and all compounds that will be reported in the future. To be specific, the aforementioned FK228, a salt thereof and a derivative thereof (e.g., acetylated FK228, a Zo thiol form wherein S-S bond has been reduced etc.) can be mentioned. In addition, trichostatin A, sodium butyrate, suberoylanilide hydroxamic acid (SAHA), MS-275, Cyclic hydroxamic-acid-containing peptide, Apicidin, Trapoxin and the like are also compounds whose histone deacetylase inhibitory 15 activity has been reported.
The evaluation method of an antitumor effect of the histone deacetylase inhibitor of the present invention includes at least (i) a step of treating a test cell with a histone deacetylase inhibitor, and (ii) a step of measuring 2o change in the expression of a specific gene and/or a protein in the test cell before and after the treatment with said inhibitor, and comparing the both expression amounts. Each step is explained in detail in the following.
(i) Step of treating a test cell with a histone deacetylase 2s inhibitor In this step, a test cell is cultured in a solution containing a histone deacetylase inhibitor.
While the test cell to be used in the present invention is not particularly limited as long as it has histone 3o deacetylase, since evaluation of an antitumor effect of the histone deacetylase inhibitor, particularly tumor site specificity of the inhibitor, is one of the problems of the present invention, the test cell to be used is preferably DOCSTOR: 639063\1 derived from a tumor on which the effect is desired to be examined. For example, when the effect on prostate cancer is to be evaluated, PC-3 cell, which is a cultured human prostate cancer cell, and the like are used, and when the effect on s kidney cancer is to be evaluated, ACHN cell, which is a cultured human kidney cancer cell and the like are used.
Various cultured human cancer cells to be used as test cells including these cancer cells are commercially available, or available from various cell banks and the like. For to examination of a long-term treatment effect, or effectiveness for individual patients, namely, tailor made medicine, it is possible to culture a cancer cell that can be obtained from a tumor of patient and use the cancer cell as a test cell.
The histone deacetylase inhibitor to be used for this 1s step is as mentioned above.
The treatment conditions of the test cell and the histone deacetylase inhibitor are free of any particular limitation as long as the effect of the histone deacetylase inhibitor can be fully exerted, and appropriately set 2o according to the factors such as the kind of the test cell to be used and the kind of histone deacetylase inhibitor to be tested and the like.
The solvent to give a solution of a histone deacetylase inhibitor is not particularly limited as long as it can 2s dissolve the histone deacetylase inhibitor and it does not show toxicity to the test cell. Generally, a concentrated solution is prepared with ethanol, PEG400, loo HCO-60 solution, dimethyl sulfoxide and the like, a mixed solvent thereof and the like, and diluted to a desired concentration with a 3o culture medium, physiological buffer and the like and used.
The concentration of the histone deacetylase inhibitor in the solution is generally 0.001-1000 nM, preferably 0.01-100 nM, more preferably 0.1-10 nM, and in some cases, the solution is DOCSTOR: 639063\1 serially diluted and a serial dilution series is made and used.
In the evaluation method of the present invention, the number of test cells to be inoculated can be appropriately increased or decreased depending on the treatment time and the s like with a histone deacetylase inhibitor. It is generally about 1x103-1x106 cells, preferably about 1x104-1x105 cells, per 1 mL of culture medium.
The treatment time (culture time) of the test cell with a histone deacetylase inhibitor is appropriately set according to to the kind and concentration of the test cell and the inhibitor and other culture conditions, and varies depending on the object of evaluation, but it is generally 1-100 hr, preferably 1-72 hr. When confirmation of a long-term sustained antitumor effect is desired, a comparatively longer treatment Is time is set. The test cell is generally treated (cultured) at 37°C in the presence of 5o COZ+95% O2.
(ii) Step of measuring change in the expression of a specific gene and/or a specific protein in the test cell before and after the treatment with said inhibitor, and comparing the 2o both expression amounts This step can be carried out by any method by which the expression amount of a specific gene andlor a specific protein in a test cell can be observed. For example, procedures described in the following can be mentioned.
2s (1) A gene, particularly mRNA, or protein is extracted from a test cell before treatment with a histone deacetylase inhibitor.
(2) As described in detail under the above-mentioned (i) Step of treating a test cell with a histone deacetylase inhibitor, 3o after the test cell is treated with the histone deacetylase inhibitor and cultured for a given period of time, a gene, particularly mRNA, or protein is extracted from the treated cell in the same manner as in the above-mentioned (1).
DOCSTOR: 639063\ 1 (3) Using a substance having specific affinity for a specific gene (or specific protein), the specific gene (or specific protein) is detected. Here, the specific gene (or specific protein) means one that shows change in its expression amount s before and after the treatment with the histone deacetylase inhibitor and shows a correlation between the change in the expression amount and the antitumor effect of the histone deacetylase inhibitor. Specifically, p21 gene (protein) and c-myc gene (protein) can be mentioned. The p21 gene is a cell to cycle regulating gene involved in the suppression of the cell cycle progress, and its product is known to inhibit the activity of cyclin/cyclin dependent kinase complex, thereby blocking the cell cycle progress. The c-myc gene encodes an intranuclear protein and its gene expression remarkably Zs changes according to cell growth, cell development and canceration. Accordingly, involvement of the gene product in cell growth is attracting attention.
For a specific gene (or specific protein) to be measured in the present invention, one kind of measurement is zo sufficiently useful, but when a more detailed antitumor effect needs to be known, two or more kinds of specific genes (or specific proteins) are preferably measured simultaneously.
A substance having specific affinity for the specific gene or specific protein is free of any particular limitation zs as long as it has such a sensitivity as allows detection of expression in the test cells. As used herein, by the "specific affinity" is meant a property to hybridize or bind solely to an object gene or protein. As the substance to detect the specific gene, a substance completely complement to the whole 30 or a part of said gene, or a substance containing one to several mismatches within the extent satisfying the above-mentioned property can be mentioned. Specific examples include oligo- or poly-nucleotide containing a part or the entirety of DOCSTOR: 639063\1 the base sequence of the gene and complementary sequences thereof, and the like, and an appropriate substance is selected depending on the form of the gene to be detected. The derivation of the substance is not particularly limited as s long as it has specific affinity for the gene, and it may be synthesized or formed by cleaving a necessary part from the gene and purifying the part by a conventional method. The substance may be labeled with a fluorescent substance, an enzyme, a radioisotope and the like. As the substance to be Io used for detecting a specific protein, for example, an antibody having specific affinity for the protein or a fragment thereof can be mentioned. The specific affinity thereof means an ability to specifically recognize the protein by an antigen-antibody reaction and bind thereto. The antibody 15 and the fragment thereof are not particularly limited as long as they can specifically bind to the protein, and may be any of a polyclonal antibody, a monoclonal antibody and functional fragments thereof. These antibodies and functional fragments thereof can be produced according to a method generally 2o employed in the pertinent field. These antibodies and fragments thereof may be labeled with a fluorescent substance, an enzyme, a radioisotope and the like.
Extraction of a gene, particularly mRNA, as well as extraction of a protein from the test cell can be performed 2s according to a method generally employed in the pertinent field, or by an appropriate combination of such methods. When mRNA was extracted, its expression is examined according to a method generally employed in the pertinent field, such as Northern blot, RT-PCR and the like, using a substance having 3o specific affinity for the above-mentioned specific gene. On the other hand, when a protein was extracted, its expression is examined according to a method generally employed in the pertinent field, such as immunoblot, Western blot and the like, DOCSTOR: 639063\1 using a substance (antibody, a fragment thereof etc.) having specific affinity for the above-mentioned specific protein.
In this way, changes in the expression of a specific gene (or specific protein) in a test cell before and after s treatment with a histone deacetylase inhibitor is measured and compared to determine whether or not the tested histone deacetylase inhibitor has effectively shown an antitumor activity in the tested cell. When p21 gene (or protein) is used as an index and the treatment with a histone deacetylase io inhibitor increases the expression amount, the inhibitor is determined to have an antitumor effect against a tumor the test cell derived from. When c-myc gene (or protein) is used as an index and the treatment with a histone deacetylase inhibitor decreases the expression amount, the inhibitor is 15 determined to have an antitumor effect against a tumor the test cell derived from. When a tumor cell clinically obtained from a patient is used as a test cell, prediction of antitumor effect reflecting the individual specificity of patient is attainable.
2o In the present invention, a screening method of a histone deacetylase inhibitor having a tumor site (kind) specific antitumor activity can be provided by utilizing the aforementioned evaluation method of an antitumor effect of the histone deacetylase inhibitor. The tumor site specificity of 2s each inhibitor can be determined by using a test cell derived from a target tumor, treating with a histone deacetylase inhibitor whose effect is to be examined, and determining the presence or otherwise of the antitumor effect according to the aforementioned method.
3o The present invention moreover provides a method for obtaining a gene to be an index for predicting the efficacy of FK228. By analyzing the expression of the gene (group) obtained by such method, the information of whether or not DOCSTOR: 639063\ 1 FK228 is useful for the treatment, whether or not the target cancer is affected by FK228 and the like can be obtained, which can contribute to the "tailor made medicine".
The method is specifically performed as follows.
s (1) Step of treating FK228 sensitive tumor cell and FK228 resistant tumor cell with FK228 The FK228 sensitive tumor cell here means a tumor cell of the type FK228 suppresses its growth. For example, prostate cancer cell PC-3 can be mentioned as shown in the Examples to described below. In addition, SC-6, which is a gastric cancer cell, is one kind of the FK228 sensitive tumor cells. On the other hand, the FK228 resistant tumor cell is a tumor cell of the type FK228 fails to exhibit suppression of its growth and FK228 cannot provide a tumor suppression effect thereon. For is example, kidney cancer cell ACHN can be mentioned as shown in the Examples described below. Moreover, A498, which is a kidney cancer cell, is one kind of the FK228 resistant tumor cells.
The treatment of these tumor cells with FK228 is 2o conducted in the same manner as in the above-mentioned "Step of treating a test cell with a histone deacetylase inhibitor".
(2) Step of selecting genes that show increased or decreased expression by the treatment of step (1) above This step of selecting genes can be performed using the 2s techniques described in the present specification and methods generally employed in the pertinent field. A technique using a gene chip is preferably employed in view of the advantage of possible analysis of a large amount of gene expression at one time.
30 (3) Step of selecting, from the genes selected in step (2) above, (i) a gene that shows increased expression due to the treatment with FK228, higher expression in the FK228 DOCSTOR: 639063\1 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, (ii) a gene that shows increased expression due to the treatment with FK228, lower expression in the FK228 s sensitive tumor cell and higher expression in the FK228 resistant tumor cell, (iii) a gene that shows decreased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 to resistant tumor cell, or (iv) a gene that shows decreased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell.
Zs In other words, this step intends selection of genes that show some changes in the expression (increase or decrease) due to the treatment with FK228 and show a different expression state depending on whether it is sensitive or insensitive to FK228. Analyzing the state of expression of the gene (group) 2o can be a useful means for predicting the efficacy of FK228 without administration of FK228.
The method of finding increase or decrease of gene expression can be performed according to methods generally employed in the pertinent field and is performed using the 25 techniques also described in the present specification. A
technique using a gene chip is preferably employed in view of the advantage of possible analysis of a large amount of gene expression at one time.
Examples 3o The present invention is explained specifically and in detail in the following by referring to Examples, which are not to be construed as limitative.
2s (15) A method for obtaining a gene capable of becoming an index for predicting the efficacy of FK228, which comprises at least (1) a step of treating an FK228 sensitive tumor cell and an FK228 resistant tumor cell with FK228, 30 (2) a step of selecting genes that show increased or decreased expression in step (1) above, and (3) a step of selecting, from the genes selected in step (2) above, DOCSTOR: 639063\ 1 (i) a gene that shows increased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, s (ii) a gene that shows increased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell, (iii) a gene that shows decreased expression due to the to treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, or (iv) a gene that shows decreased expression due to the treatment with FK228, lower expression in the FK228 Is sensitive tumor cell and higher expression in the FK228 resistant tumor cell.
Brief Description of the Drav~rings Fig. 1 is a graph showing an antitumor effect of FR901228 on human prostate cancer, wherein the vertical axis 2o shows a tumor growth rate, the transverse axis shows the number of days lapsed from the initial administration, and the tumor growth rate is expressed in a relative proportion of tumor volume after day 0 relative to the tumor volume on day 0 taken as 1.
2s Fig. 2 includes graphs showing an antitumor effect of FR901228 on human lymphoma, wherein the vertical axis shows the proportion of survived mice, and the transverse axis shows the number of days lapsed after tumor cell implantation.
so Fig. 3 includes graphs showing an antitumor effect of FR901228 on human prostate cancer ((a); PC-3) and kidney cancer ((b): ACHN), wherein the vertical axis shows a tumor growth rate, the transverse axis shows the number of days DOCSTOR: 639063\1 lapsed after the initial administration, and the tumor growth rate is expressed in a relative proportion of tumor volume after day 0 relative to the tumor volume on day 0 taken as 1.
s Fig. 4 includes graphs showing an action of FR901228 on p21 gene expression in vitro (PC-3 cell, ACHN cell).
(a),(b); The vertical axis shows a relative amount of p21 gene expression, and the transverse axis shows contact time (hr) with FR901228.
to (c); The vertical axis shows a relative amount of p21 gene expression.
Fig. 5 includes graphs showing an action of FR901228 on p21 gene expression and c-myc gene expression in vivo (PC-3 cell, ACHN cell), wherein the vertical axis shows a 15 relative amount of p21 or c-myc gene expression, and the transverse axis shows the number of days lapsed after administration of FR901228.
Detailed Description of the Invention The therapeutic agent for prostate cancer and the 2o therapeutic agent for malignant lymphoma of the present invention comprise, as an active ingredient, a compound (FK228) represented by the formula (I) or a salt thereof. Of the compounds of the formula (I), a compound (FR901228) represented by the formula (II), which is a stereoisomer, is 25 preferable. These compounds have a strong histone deacetylase inhibitory activity (Nakajima, H. et al; ibid.
(1998)), and FR901228 is particularly preferably contained in the therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present 3o invention, because it has a stronger histone deacetylase inhibitory activity.
In the present specification, a simple reference to FK228 means a group of compounds regardless of stereoisomerism, DOCSTOR: 639063\1 including the compounds represented by the formula (II), unless otherwise specified.
FK228 and a salt thereof are known and available substances. For example, FR901228, which is one of the s stereoisomers of FK228, can be obtained by culturing a strain capable of producing FR901228 and belonging to the genus Chromobacterium under aerobic conditions and recovering the substance from the culture broth. As the strain capable of producing FR901228 and belonging to the genus Chromobacterium, io for example, Chromobacterium violaceum WB968 (FERM BP-1968) can be mentioned. More specifically, FR901228 can be obtained from a FR901228-producing strain according to the method described in JP-B-7-64872 (corresponding to US Patent No.
4977138). FR901228 is preferably recovered from a strain i5 capable of producing FR901228 and belonging to the genus Chromobacterium, because it is obtained more easily. However, synthetic or semi-synthetic FR901228 is also advantageous, because no or only fewer steps of purification is/are required.
Likewise, FK228 other than FR901228 can be also semi-ao synthesized or totally synthesized according to a conventionally known method. More specifically, it can be produced according to the method reported by Khan W.Li, et al (J. Am. Chem. Soc., vol. 118, 7237-7238 (1996)).
The salt of FK228 is a biologically acceptable, generally 2s nontoxic salt, and examples thereof include salts with inorganic base (e. g., alkali metal salts such as sodium salt, potassium salt etc., alkaline earth metal salts such as calcium salt, magnesium salt etc., and ammonium salt), salts with organic base (e.g., organic amine salts such as 3o triethylamine salt, diisopropylethyl amine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt etc.), inorganic acid addition salts (e. g., hydrochloride, DOCSTOR: 639063\1 hydrobromide, hydrosulfate, phosphate etc.), organic carboxylic acid ~ sulfonic acid addition salts (e. g., formate, acetate, trifluoroacetate, maleate, tartrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate etc.), s salts with a basic or acidic amino acid (e. g., arginine, aspartic acid, glutamic acid etc.), salts with a base and acid addition salts.
FK228 may have a stereoisomer (e.g., FR901228) such as an optical isomer or geometric isomer based on an asymmetric ~o carbon atom or a double bond, and all the isomers and a mixture thereof are within the scope of the present invention.
Solvate compounds of FK228, FR901228 and salts thereof (e. g., inclusion compounds (e. g., hydrate etc.)) are also encompassed in the scope of the present invention.
Is In the present invention, in vivo and in vitro generally mean as used in the pertinent field. That is, "in vivo" refers to a state where an object biological function or reaction is expressed in the living organism, and "in vitro" refers to an expression of such function or reaction in a test tube (tissue 2o culture system, cell culture system, cell free system etc.).
The tumor to be the target in the present invention is a tumor on which FK228, which is a histone deacetylase inhibitor, exerts an antitumor effect, and examples thereof include prostate cancer and malignant lymphoma, where in vivo effect 2s is particularly remarkable. The malignant lymphoma, on which the therapeutic agent for malignant lymphoma of the present invention shows an antitumor effect, is preferably that other than T cell lymphoma, such as B cell lymphoma, histiocytosis lymphoma and the like. The present invention shows fine 3o antitumor effect in vivo particularly against these tumors.
The therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present invention can be used as a pharmaceutical preparation in the DOCSTOR: 639063\1 form of a solid, semi-solid or liquid containing FK228 or a salt thereof as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for oral or parenteral application. The active ingredient can be admixed s with a conventional, nontoxic, pharmaceutically acceptable carrier for, for example, powder, tablet, pellet, capsule, suppository, liquid, emulsion, suspension, aerosol, spray and other form suitable for use. Where necessary, auxiliary, stabilizer, thickening agent and the like may be also used.
so These carriers and excipients may be used after a sterilization treatment where necessary, or may be sterilized after formulation into a preparation. FK228 or a salt thereof may be contained in an amount sufficient to provide an antitumor effect, in the therapeutic agent for prostate cancer Is and the therapeutic agent for malignant lymphoma.
When the pharmaceutical agent is applied to a human, it is preferably applied by intravenous, intramuscular or oral administration. While the therapeutically effective dose of FK228 or a salt thereof, which are active ingredients, varies 2o depending on the age and condition of individual patients to be treated, and the kind of cancer and the kind of malignant lymphoma, it is generally 0.1-100 mg, preferably 1-50 mg, more preferably 5-30 mg, a day in the amount of FK228 per human body surface area (m2) in the case of intravenous 2s administration for the treatment of tumor.
The present invention also provides an evaluation method of an antitumor effect of the histone deacetylase inhibitor.
Using this method, a histone deacetylase inhibitor that can exert an antitumor effect on the target tumor cell can be 3o found without actually administering the inhibitor to a human body.
By the "histone deacetylase inhibitor" is meant a compound that binds to the active site of histone deacetylase DOCSTOR: 639063\1 competitively with substrate, or a compound that binds to a site other than the active site of histone deacetylase and has an action to alter the enzyme activity of histone deacetylase, and it encompasses a compound already known as a histone s deacetylase inhibitor whose use is known, all compounds (synthetic or natural) reported to have a histone deacetylase inhibitory activity and all compounds that will be reported in the future. To be specific, the aforementioned FK228, a salt thereof and a derivative thereof (e.g., acetylated FK228, a Zo thiol form wherein S-S bond has been reduced etc.) can be mentioned. In addition, trichostatin A, sodium butyrate, suberoylanilide hydroxamic acid (SAHA), MS-275, Cyclic hydroxamic-acid-containing peptide, Apicidin, Trapoxin and the like are also compounds whose histone deacetylase inhibitory 15 activity has been reported.
The evaluation method of an antitumor effect of the histone deacetylase inhibitor of the present invention includes at least (i) a step of treating a test cell with a histone deacetylase inhibitor, and (ii) a step of measuring 2o change in the expression of a specific gene and/or a protein in the test cell before and after the treatment with said inhibitor, and comparing the both expression amounts. Each step is explained in detail in the following.
(i) Step of treating a test cell with a histone deacetylase 2s inhibitor In this step, a test cell is cultured in a solution containing a histone deacetylase inhibitor.
While the test cell to be used in the present invention is not particularly limited as long as it has histone 3o deacetylase, since evaluation of an antitumor effect of the histone deacetylase inhibitor, particularly tumor site specificity of the inhibitor, is one of the problems of the present invention, the test cell to be used is preferably DOCSTOR: 639063\1 derived from a tumor on which the effect is desired to be examined. For example, when the effect on prostate cancer is to be evaluated, PC-3 cell, which is a cultured human prostate cancer cell, and the like are used, and when the effect on s kidney cancer is to be evaluated, ACHN cell, which is a cultured human kidney cancer cell and the like are used.
Various cultured human cancer cells to be used as test cells including these cancer cells are commercially available, or available from various cell banks and the like. For to examination of a long-term treatment effect, or effectiveness for individual patients, namely, tailor made medicine, it is possible to culture a cancer cell that can be obtained from a tumor of patient and use the cancer cell as a test cell.
The histone deacetylase inhibitor to be used for this 1s step is as mentioned above.
The treatment conditions of the test cell and the histone deacetylase inhibitor are free of any particular limitation as long as the effect of the histone deacetylase inhibitor can be fully exerted, and appropriately set 2o according to the factors such as the kind of the test cell to be used and the kind of histone deacetylase inhibitor to be tested and the like.
The solvent to give a solution of a histone deacetylase inhibitor is not particularly limited as long as it can 2s dissolve the histone deacetylase inhibitor and it does not show toxicity to the test cell. Generally, a concentrated solution is prepared with ethanol, PEG400, loo HCO-60 solution, dimethyl sulfoxide and the like, a mixed solvent thereof and the like, and diluted to a desired concentration with a 3o culture medium, physiological buffer and the like and used.
The concentration of the histone deacetylase inhibitor in the solution is generally 0.001-1000 nM, preferably 0.01-100 nM, more preferably 0.1-10 nM, and in some cases, the solution is DOCSTOR: 639063\1 serially diluted and a serial dilution series is made and used.
In the evaluation method of the present invention, the number of test cells to be inoculated can be appropriately increased or decreased depending on the treatment time and the s like with a histone deacetylase inhibitor. It is generally about 1x103-1x106 cells, preferably about 1x104-1x105 cells, per 1 mL of culture medium.
The treatment time (culture time) of the test cell with a histone deacetylase inhibitor is appropriately set according to to the kind and concentration of the test cell and the inhibitor and other culture conditions, and varies depending on the object of evaluation, but it is generally 1-100 hr, preferably 1-72 hr. When confirmation of a long-term sustained antitumor effect is desired, a comparatively longer treatment Is time is set. The test cell is generally treated (cultured) at 37°C in the presence of 5o COZ+95% O2.
(ii) Step of measuring change in the expression of a specific gene and/or a specific protein in the test cell before and after the treatment with said inhibitor, and comparing the 2o both expression amounts This step can be carried out by any method by which the expression amount of a specific gene andlor a specific protein in a test cell can be observed. For example, procedures described in the following can be mentioned.
2s (1) A gene, particularly mRNA, or protein is extracted from a test cell before treatment with a histone deacetylase inhibitor.
(2) As described in detail under the above-mentioned (i) Step of treating a test cell with a histone deacetylase inhibitor, 3o after the test cell is treated with the histone deacetylase inhibitor and cultured for a given period of time, a gene, particularly mRNA, or protein is extracted from the treated cell in the same manner as in the above-mentioned (1).
DOCSTOR: 639063\ 1 (3) Using a substance having specific affinity for a specific gene (or specific protein), the specific gene (or specific protein) is detected. Here, the specific gene (or specific protein) means one that shows change in its expression amount s before and after the treatment with the histone deacetylase inhibitor and shows a correlation between the change in the expression amount and the antitumor effect of the histone deacetylase inhibitor. Specifically, p21 gene (protein) and c-myc gene (protein) can be mentioned. The p21 gene is a cell to cycle regulating gene involved in the suppression of the cell cycle progress, and its product is known to inhibit the activity of cyclin/cyclin dependent kinase complex, thereby blocking the cell cycle progress. The c-myc gene encodes an intranuclear protein and its gene expression remarkably Zs changes according to cell growth, cell development and canceration. Accordingly, involvement of the gene product in cell growth is attracting attention.
For a specific gene (or specific protein) to be measured in the present invention, one kind of measurement is zo sufficiently useful, but when a more detailed antitumor effect needs to be known, two or more kinds of specific genes (or specific proteins) are preferably measured simultaneously.
A substance having specific affinity for the specific gene or specific protein is free of any particular limitation zs as long as it has such a sensitivity as allows detection of expression in the test cells. As used herein, by the "specific affinity" is meant a property to hybridize or bind solely to an object gene or protein. As the substance to detect the specific gene, a substance completely complement to the whole 30 or a part of said gene, or a substance containing one to several mismatches within the extent satisfying the above-mentioned property can be mentioned. Specific examples include oligo- or poly-nucleotide containing a part or the entirety of DOCSTOR: 639063\1 the base sequence of the gene and complementary sequences thereof, and the like, and an appropriate substance is selected depending on the form of the gene to be detected. The derivation of the substance is not particularly limited as s long as it has specific affinity for the gene, and it may be synthesized or formed by cleaving a necessary part from the gene and purifying the part by a conventional method. The substance may be labeled with a fluorescent substance, an enzyme, a radioisotope and the like. As the substance to be Io used for detecting a specific protein, for example, an antibody having specific affinity for the protein or a fragment thereof can be mentioned. The specific affinity thereof means an ability to specifically recognize the protein by an antigen-antibody reaction and bind thereto. The antibody 15 and the fragment thereof are not particularly limited as long as they can specifically bind to the protein, and may be any of a polyclonal antibody, a monoclonal antibody and functional fragments thereof. These antibodies and functional fragments thereof can be produced according to a method generally 2o employed in the pertinent field. These antibodies and fragments thereof may be labeled with a fluorescent substance, an enzyme, a radioisotope and the like.
Extraction of a gene, particularly mRNA, as well as extraction of a protein from the test cell can be performed 2s according to a method generally employed in the pertinent field, or by an appropriate combination of such methods. When mRNA was extracted, its expression is examined according to a method generally employed in the pertinent field, such as Northern blot, RT-PCR and the like, using a substance having 3o specific affinity for the above-mentioned specific gene. On the other hand, when a protein was extracted, its expression is examined according to a method generally employed in the pertinent field, such as immunoblot, Western blot and the like, DOCSTOR: 639063\1 using a substance (antibody, a fragment thereof etc.) having specific affinity for the above-mentioned specific protein.
In this way, changes in the expression of a specific gene (or specific protein) in a test cell before and after s treatment with a histone deacetylase inhibitor is measured and compared to determine whether or not the tested histone deacetylase inhibitor has effectively shown an antitumor activity in the tested cell. When p21 gene (or protein) is used as an index and the treatment with a histone deacetylase io inhibitor increases the expression amount, the inhibitor is determined to have an antitumor effect against a tumor the test cell derived from. When c-myc gene (or protein) is used as an index and the treatment with a histone deacetylase inhibitor decreases the expression amount, the inhibitor is 15 determined to have an antitumor effect against a tumor the test cell derived from. When a tumor cell clinically obtained from a patient is used as a test cell, prediction of antitumor effect reflecting the individual specificity of patient is attainable.
2o In the present invention, a screening method of a histone deacetylase inhibitor having a tumor site (kind) specific antitumor activity can be provided by utilizing the aforementioned evaluation method of an antitumor effect of the histone deacetylase inhibitor. The tumor site specificity of 2s each inhibitor can be determined by using a test cell derived from a target tumor, treating with a histone deacetylase inhibitor whose effect is to be examined, and determining the presence or otherwise of the antitumor effect according to the aforementioned method.
3o The present invention moreover provides a method for obtaining a gene to be an index for predicting the efficacy of FK228. By analyzing the expression of the gene (group) obtained by such method, the information of whether or not DOCSTOR: 639063\ 1 FK228 is useful for the treatment, whether or not the target cancer is affected by FK228 and the like can be obtained, which can contribute to the "tailor made medicine".
The method is specifically performed as follows.
s (1) Step of treating FK228 sensitive tumor cell and FK228 resistant tumor cell with FK228 The FK228 sensitive tumor cell here means a tumor cell of the type FK228 suppresses its growth. For example, prostate cancer cell PC-3 can be mentioned as shown in the Examples to described below. In addition, SC-6, which is a gastric cancer cell, is one kind of the FK228 sensitive tumor cells. On the other hand, the FK228 resistant tumor cell is a tumor cell of the type FK228 fails to exhibit suppression of its growth and FK228 cannot provide a tumor suppression effect thereon. For is example, kidney cancer cell ACHN can be mentioned as shown in the Examples described below. Moreover, A498, which is a kidney cancer cell, is one kind of the FK228 resistant tumor cells.
The treatment of these tumor cells with FK228 is 2o conducted in the same manner as in the above-mentioned "Step of treating a test cell with a histone deacetylase inhibitor".
(2) Step of selecting genes that show increased or decreased expression by the treatment of step (1) above This step of selecting genes can be performed using the 2s techniques described in the present specification and methods generally employed in the pertinent field. A technique using a gene chip is preferably employed in view of the advantage of possible analysis of a large amount of gene expression at one time.
30 (3) Step of selecting, from the genes selected in step (2) above, (i) a gene that shows increased expression due to the treatment with FK228, higher expression in the FK228 DOCSTOR: 639063\1 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, (ii) a gene that shows increased expression due to the treatment with FK228, lower expression in the FK228 s sensitive tumor cell and higher expression in the FK228 resistant tumor cell, (iii) a gene that shows decreased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 to resistant tumor cell, or (iv) a gene that shows decreased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell.
Zs In other words, this step intends selection of genes that show some changes in the expression (increase or decrease) due to the treatment with FK228 and show a different expression state depending on whether it is sensitive or insensitive to FK228. Analyzing the state of expression of the gene (group) 2o can be a useful means for predicting the efficacy of FK228 without administration of FK228.
The method of finding increase or decrease of gene expression can be performed according to methods generally employed in the pertinent field and is performed using the 25 techniques also described in the present specification. A
technique using a gene chip is preferably employed in view of the advantage of possible analysis of a large amount of gene expression at one time.
Examples 3o The present invention is explained specifically and in detail in the following by referring to Examples, which are not to be construed as limitative.
DOCSTOR: 639063\I
Example 1 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent (10% HCO-60/saline) was added. The mixture was sonicated to s allow for dissolution. A positive control substance Paclitaxel was dissolved in Cremophor EL/ethanol (1:1) solution to 24 mg/mL prior to the testing, and preserved in a refrigerator.
When in use, it was diluted with a 9-fold amount of physiological saline to 2.4 mg/mL (solvent component: 5%
to Cremophor EL-5% ethanol-90% saline).
(2) Test animal For antitumor test of the pharmaceutical agent, BALB/cANnNCrj-nu/nu mice (male, 6-week-old) were purchased from Charles River Japan and, after acclimation for not less Is than one week, used for the test. The mice were reared under an SPF environment and allowed a free access to water and feed.
(3) Test tumor Cultured human prostate cancer cell line (PC-3: available from Japanese Foundation for Cancer Research, Cancer Zo Chemotherapy Center, Fundamental Research) was subcutaneously implanted at 2-3x10' cells in a nude mouse. A grown solid tumor was subcultured not less than 3 generations and used for the test.
(4) Experimental implantation and grouping 2s A solid tumor subcultured in a nude mouse was subcutaneously implanted in the right back of a mouse as an about 3 mm square tumor tissue fragment. After the tumor implantation, when the tumor volume (1/2xlonger diameterxshorter diameter2) reached 100-300 mm3, the mice were 3o grouped into 6 mice per group to level the tumor size.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intravenously administered to an DOCSTOR: 639063\ l FR901228 administration group 3 times every 4 days (q4dx3) (3.2 and 1.8 mg/kg). Paclitaxel (24 mg/kg) was intravenously administered for 5 consecutive days (qdx5) to a positive control substance paclitaxel administration group. Only a s solvent (loo HCO-60/saline) was administered (q4dx3) to a control group. The amount of liquid for each administration was calculated (0.1 mL/10 g body weight) based on the body weight measured on the administration day. Note that 3.2 mg/kg/day (q4dx3) of FR901228 and 24 mg/kg/day (qdx5) of to Paclitaxel were the maximum tolerated doses (MTD) thereof.
(6) Measurement of tumor size and body weight The tumor size (longer diameter, shorter diameter) and body weight were measured twice a week from Day 0.
(7) Evaluation of antitumor effect is The level of tumor growth was evaluated based on the tumor growth rate (Relative Tumor Volume). The growth suppression rate was expressed in a relative proportion of tumor volume after day 0 to the tumor volume at Day 0 as 1.
The antitumor effect was determined on the 14th day (Day 14) 2o from the start of the administration of the pharmaceutical agent. When the proportion of the tumor growth rate of the pharmaceutical agent administration group to that of the control group (solvent administration), (T/C%), was not more than 50o and a significant difference (P<0.01) was found in zs the Mann Whitney U-test, the pharmaceutical agent was determined to be effective.
The results are shown in Fig. 1. FR901228 showed an antitumor effect in vivo against human prostate cancer.
Example 2 30 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent (10s HCO-60/saline) was added. The mixture was sonicated to allow for dissolution.
DOCSTOR: 639063\I
(2) Test animal For antitumor test of the pharmaceutical agent, Fox Chase C.B-17/Icr-SCID.Jcl mice (male, 6-week-old) were purchased from CLEA JAPAN INC. and, after acclimation for not less than s one week, used for the test. The mice were reared under an SPF
environment and allowed a free access to water and feed.
(3) Test tumor Cultured human lymphoma cell line (U937: obtained from Dr.
Minowada, Hayashibara Biochemical Laboratories, Inc.) was to cultured in RPMI (containing 10% FCS) and subcultured in vitro.
(4) Experimental implantation and grouping Cyclo phosphamide (Shionogi & Co., Ltd., 150 mg/kg) was intraperitoneally administered to mice. Lymphoma (1x107 cells) subcultured in vitro was intraperitoneally implanted the next is day. After the tumor implantation, the mice were grouped into 6 (control group 12) mice per group to level the body weight.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intraperitoneally administered to an 2o FR901228 administration group once or twice a week (0.1-1.0 mg/kg). Only a solvent (loo HCO-60/saline) was administered to a control group.
(6) Evaluation As an antitumor effect, the survival days of the mice 2s were counted.
The results are shown in Fig. 2. Fig. 2(a) shows the results of administration of FR901228 once a week, and Fig.
2(b) shows the results of administration of FR901228 twice a week. FR901228 showed an antitumor effect in vivo against 3o human lymphoma.
Example 3 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent DOCSTOR: 639063\1 (10% HCO-60/saline) was added. The mixture was sonicated to allow for dissolution. A positive control substance Paclitaxel was dissolved in Cremophor EL/ethanol (1:1) solution to 24 mg/mL prior to the testing, and preserved in a refrigerator.
s When in use, it was diluted with a 9-fold amount of physiological saline to 2.4 mg/mL (solvent component: 50 Cremophor EL-5o ethanol-90o saline).
(2) Test animal For antitumor test of the pharmaceutical agent, to BALB/cANnNCrj-nu/nu mice (male, 6-week-old) were purchased from Charles River Japan and, after acclimation for not less than one week, used for the test. The mice were reared under an SPF environment and allowed a free access to water and feed.
(3) Test tumor is Cultured human kidney cancer cell line 1 (ACHN: available from ATCC) and cultured human prostate cancer cell line 1 (PC-3: available from ATCC) were subcutaneously implanted at 2-3x10' cells in a nude mouse. A grown solid tumor was subcultured not less than 3 generations and used for the test.
2o (4) Experimental implantation and grouping A solid tumor subcultured in a nude mouse was subcutaneously implanted into the right back of a mouse as an about 3 mm square tumor tissue fragment. After the tumor implantation, when the tumor volume (1/2xlonger 2s diameterxshorter diameter2) reached 100-300 mm3, the mice were grouped into 6 mice per group to level the tumor size.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intravenously administered to an 3o FR901228 administration group 3 times every 4 days (q4dx3) (3.2 and 1.8 mg/kg). Paclitaxel was intravenously administered (24 mg/kg) for 5 consecutive days (qdx5) to a positive control substance Paclitaxel administration group. Only a solvent (10%
DOCSTOR: 639063\1 HCO-60/saline) was administered (q4dx3) to a control group.
The amount of liquid for each administration was calculated (0.1 mL/10 g body weight) based on the body weight measured on the administration day. Note that 3.2 mg/kg/day (q4dx3) of s FR901228 and 24 mg/kg/day (qdXS) of paclitaxel were MTDs thereof .
(6) Measurement of tumor size and body weight The tumor size (longer diameter, shorter diameter) and body weight were measured twice a week from Day 0.
to (7) Evaluation of antitumor effect The level of tumor growth was evaluated based on the tumor growth rate (Relative Tumor Volume). The growth suppression rate was expressed in a relative proportion of tumor volume after day 0 to the tumor volume at Day 0 as 1.
Is The antitumor effect was determined on the 14th day (Day 14) from the start of the administration of a pharmaceutical agent.
When the ratio of the pharmaceutical agent administration group to the tumor growth rate of the control group (solvent administration) (T/Co) was not more than 500, and a zo significant difference (P<0.01) was found in the Mann Whitney U-test, the pharmaceutical agent was determined to be effective.
The results are shown in Fig. 3. FR901228 showed a potent antitumor effect against PC-3 at the dose of 3.2 mg/kg 2s (Fig. 3(a)) but did not show an antitumor effect against ACHN
(Fig. 3(b)).
Example 4 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and dissolved 3o in a solvent (99.50 ethanol) to the concentration of 1 mg/mL.
Then, the solution was diluted with culture medium.
(2) Test tumor Cultured human cancer cell (PC-3 and ACHN) were cultured DOCSTOR: 639063\1 in DMEM (containing loo FCS).
(3) Culture and RNA extraction The cells were inoculated at 2x106 cells per a culture dish, and cultured in the presence of FR901228 (5 ng/mL) for a s given time. After culturing, RNA was extracted with a TRIZOL
reagent (GIBCO BRL) according to the operation manual.
(4) Real time PCR
RNA was subjected to reverse transcription using a Taq man reverse transcription reagent (PE Biosystem) according to io the operation manual. Thereafter, p21 gene was amplified using a SYBR green PCR master mix (PE Biosystem) and primer 5'-GGC
AGA CCA GCA TGA CAC ATT-3' (p21 upstream)(SEQ ID; No 1), 5'-GGA TTA GGG CTT CCT CTT GGA G-3' (SEQ ID; No 2) according to the operation manual, and detected with ABI 7700 PRISM
is sequence detector (PE Biosystem). The expression amount of p21 gene was calculated from a standard curve, divided by the expression amount of ~3-actin gene, which was used as an internal standard, and expressed in a standardized relative expression amount.
2o The results are shown in Fig. 4. By being contacted with FR901228 in vitro, PC-3 (Fig. 4(a)) showed an increased expression of the p21 gene with the lapse of time. In contrast, ACHN did not show increased expression of p21 gene (Fig. 4(b)).
When untreated, p21 gene showed little expression in PC-3 but 2s showed expression in ACHN (Fig. 4(c)).
Example 5 Human prostate.cancer PC-3 or kidney cancer ACHN was subcutaneously implanted in a nude mouse, and when the size of the tumor reached 100-300 mg, FR901228 (3.2 mg/kg) was 3o intravenously administered. The tumor was removed with the lapse of time, and after extracting RNA, the expression amount of p21 gene and c-myc gene was examined by real time PCR in the same manner as in Example 4. The c-myc gene was amplified DOCSTOR: 639063\1 using a SYBR green PCR master mix (PE Biosystem) and primer 5'-GAC AGA TCA GCA ACA ACC GAA A-3' (human c-myc upstream)(SEQ
ID; No 3), 5'-TTG TGT GTT CGC CTC TTG ACA T-3' (human c-myc downstream)(SEQ ID; No 4) according to the operation manual, s and detected with ABI 7700 PRISM sequence detector (PE
Biosystem).
The results are shown in Fig. 5. PC-3 showed increased expression of p21 gene in vivo with a peak at 3 hr after administration of FR901228 (Fig. 5(a)). In contrast, ACHN did 1o not show increased expression of p21 gene (Fig. 5(a)). While c-myc gene showed decreased expression in PC-3, it showed increased expression in ACHN (Fig. (b)).
Example 6: Analysis (in vitro) of gene expression by FK228 in tumor cell using a gene chip Is The effect of FK228 on in vitro gene expression in human tumor cell was analyzed using a gene chip.
<material ~ procedure>
(1) Test materials pharmaceutical agent FK228 (FR901228) 2o concentration at use: 50 ng/mL
preparation method: A 10 mg/mL solution was prepared with ethanol in advance and serially diluted with a culture medium to give 50 ng/mL solutions.
dosage form: solution (prepared when in use) 2s Cells used: human prostate cancer (PC-3), human lymphoma (U937), human kidney cancer (ACHN) Culture medium: DMEM (for PC-3, ACHN), RPMI1640 (for U937) Both obtained from Nikken Biomedical Laboratory, further containing FCS (Moregate) and Penicillin-Streptomycin 30 (ICN Biomedicals Inc.).
RNA extraction: RNeasy Mini Kit (50) (Qiagen) RNase, DNase free water (Life Technologies) DOCSTOR: 639063\ 1 DNA synthesis: Superscript Choice System (Life Technologies) Ethachinmate (Nippon gene) T7-(dT)24 Primer (Amersham Pharmacia) cRNA synthesis: BioArray RNA Transcript Labeling Kit (Amersham s Pharmacia) cRNA fragmentation: Trizma Base (SIGMA) glacial acetic acid (SIGMA) magnesium acetate (SIGMA) potassium acetate (SIGMA) to Hybridization: Eukaryotic Hybridization Control Kit (Amersham Pharmacia) 0.5M EDTA solution (SIGMA) MES Sodium Salt (SIGMA) MES Free Acid Monohydrate (SIGMA) is Herring Sperm DNA (Promega) Acetylated Bovine Serum Albumin Soln. (Life Technologies) Staining: Phycoerythrin-Streptavidin (Molecular Probes) Goat IgG, Reagent Grade (SIGMA) 2o Anti-streptavidin ab (goat), biotinylated (Vector Lab) Chip used: HuGeneFL array (Amersham Pharmacia) (2) Cell preparation and RNA extraction Human tumor cells (PC-3, U937, ACHN) that reached zs confluent in F75 flasks were subjected to a trypsin treatment to give single cell suspensions, which were inoculated to five F75 flasks and cultured for 24 hr. The culture medium was discarded and a fresh culture medium (18 mL) and a 10-fold concentration (50 ng/mL) of an FR901228 solution (2 mL) were 3o added. The mixture was cultured in a COZ incubator at 37°C for a given time (0, 1, 3, 12 and 24 hr). After the completion of the culturing, the culture medium was discarded and total RNA
was extracted according to the protocol of RNeasy Mini Kit DOCSTOR: 639063\ 1 (50) (Qiagen). RNA was quantified and confirmed by electrophoresis.
(3) Synthesis of cRNA
According to the GeneChip manual, Chapter 2 - Chapter 4, s and the manuals of RNeasy Mini Kit and RNA Transcript Labeling Kit, RNA was purified, cDNA was synthesized, cRNA was synthesized and cRNA was fragmented.
(4) Hybridization, Washing-staining, Scanning Hybridization, washing-staining and scanning were io conducted according to the GeneChip manual Chapter 5 - Chapter 7.
(5) Analysis Analyzed using GeneSpring (microarray data analysis soft:
manufactured by Silicon Genetics).
is <Results>
Human prostate cancer PC-3 and human lymphoma U937, which are FK228 sensitive tumor cells, and human kidney cancer ACHN, which is an FK228 resistant tumor cell, were brought into contact in vitro with FK228 with the lapse of time, and RNA
2o was extracted thereafter, and 7070 genes detectable using GeneChip were examined for genes showing change in the expression due to FK228. Such genes were analyzed according to the following procedures.
Analysis 1: Selection of gene that shows change in expression 2s due to inhibition of histone deacetylase.
For limiting the gene that shows change in expression due to FK228, a gene showing linear change of expression was selected (analysis condition of GeneSpring: gene that shows change in expression by 0.5 time or more or 0.5 time or less so at any time).
Analysis 2: Selection of gene involved in efficacy When contacted with FK228 for 72 hr, growth suppression effect on PC-3, U937 and ACHN in ICSO value was 3.17, 3.20 and DOCSTOR: 639063\1 4.25 ng/mL, respectively, showing almost the same degree of growth suppressive effect on these tumor cells. From these results, the genes relating to the growth suppression were considered to commonly show change in expression in any cell.
s There were found 105 genes that commonly showed increased expression in these three kinds of human tumor cells and 100 genes that commonly showed decreased expression in these three kinds of human tumor cells.
Example 7: Analysis of gene expression (in vivo) by FK228 in Io tumor cell using a gene chip The effect of FK228 on in vivo gene expression in human tumor cell was analyzed using a gene chip.
<material ~ procedure>
(1) Test materials i5 Pharmaceutical agent FK228 (FR901228) dose: 10 mg/kg administration dose: 10 mL/kg solvent: 10% HCO-60/saline solution dosage form: solution (prepared when in use) 2o Tumor cell: human prostate cancer PC-3 (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) human gastric cancer SC-6; obtained from Central Institute for Experimental Animals (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) 2s human kidney cancer ACHN (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) human kidney cancer A498; obtained from ATCC
(tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) 3o Subcultured animal: male BALB c/nu/nu RNA extraction: RNeasy Mini Kit (50) (Qiagen) RNase, DNase free water (Life Technologies) DOCSTOR: 63906311 DNA synthesis: Superscript Choice System (Life Technologies) Ethachinmate (Nippon gene) T7-(dT)24 Primer (Amersham Pharmacia) cRNA synthesis: BioArray RNA Transcript Labeling Kit (Amersham s Pharmacia) cRNA fragmentation: Trizma Base (SIGMA) glacial acetic acid (SIGMA) magnesium acetate (SIGMA) potassium acetate (SIGMA) to Hybridization: Eukaryotic Hybridization Control Kit (Amersham Pharmacia) 0.5 M EDTA solution (SIGMA) MES Sodium Salt (SIGMA) MES Free Acid Monohydrate (SIGMA) is Herring Sperm DNA (Promega) Acetylated Bovine Serum Albumin Soln. (Life Technologies) Chip used: HuGeneFL array (Amersham Pharmacia) (2) Cell preparation and RNA extraction 20 A 3 mm square tumor fragment (PC-3, SC-6, ACHN, A498) was subcutaneously implanted in a nude mouse and, when the tumor reached about 100 mg (longer diameter 9 mm, shorter diameter 8 mm), FR901228 (10 mg/kg) was intravenously administered. At 0, 0.5, 1, 2 and 4 hr after administration of FR901228, the tumor as was removed and total RNA was extracted according to the protocol of RNeasy Mini Kit (50) (Qiagen). RNA was quantified and confirmed by electrophoresis.
(3) Synthesis of cRNA
According to the GeneChip manual, Chapter 2 - Chapter 4, 3o and the manuals of RNeasy Mini Kit and RNA Transcript Labeling Kit, RNA was purified, cDNA was synthesized, cRNA was synthesized and cRNA was fragmented.
(4) Hybridization, Washing-staining, Scanning DOCSTOR: 639063\1 Hybridization, washing-staining and scanning were conducted according to the GeneChip manual Chapter 5 - Chapter 7.
(5) Analysis s Analyzed using GeneSpring (microarray data analysis soft:
manufactured by Silicon Genetics).
<Results>
FR901228 (10 mg/kg) was intravenously administered to human prostate cancer PC-3, human gastric cancer SC-6, human to kidney cancer ACHN and human kidney cancer A498-carrying cancer mice and tumor was removed with the lapse of time (0, 0.5, 1, 2 and 4 hr). RNA was then extracted and 7070 genes detectable using GeneChip were examined for genes showing change in the expression due to FR901228.
Is The growth suppression rate of human prostate cancer PC-3, human gastric cancer SC-6, human kidney cancer ACHN and human kidney cancer A498 by the administration of FR901228 (3.2 mg/kg) was 98%, 840, 20o and 290, respectively. Therefore, PC-3 and SC-6 were determined to be FK228 sensitive tumors, and 2o ACHN and A498 were determined to be FK228 resistant tumors.
Example 8: Mode of gene expression in FK228 sensitive tumor and FK228 resistant tumor The correlation between the mode of gene expression and efficacy of FK228 was examined in the tumor confirmed to be 2s sensitive or resistant in Example 7. Close attention was given to the genes (105 genes) that showed increased expression by FK228 treatment and genes (100 genes) that showed decreased expression thereby as demonstrated in the in vitro test in Example 6, and the correlation between these genes and 3o efficacy was studied. Furthermore, a gene that showed high expression in a sensitive tumor and low expression in a resistant tumor, and a gene that showed low expression in a sensitive tumor and high expression in a resistant tumor were DOCSTOR: 6390630 searched for. As a result, of the 105 genes that showed increased expression in vitro by treatment with FR901228, 6 genes were found to show high expression in a sensitive tumor and low expression in a resistant tumor, and 4 genes were s found to show low expression in a sensitive tumor and high expression in a resistant tumor (Table 1). In addition, of the 100 genes that showed decreased expression in vitro by treatment with FR901228, 4 genes were found to show high expression in a sensitive tumor and low expression in a to resistant tumor, and 9 genes were found to show low expression in a sensitive tumor and high expression in a resistant tumor (Table 2).
Table 1: genes that showed high expression in a sensitive is tumor and low expression in a resistant tumor, or genes that showed low expression in a sensitive tumor and high expression in a resistant tumor (genes that showed increased expression in vitro by treatment with FR901228) 2o Sensitive Tumor (High) and Resistant Tumor (Low) M13686,s_at (SFTP1) pulmonary surfactant-associated protein U68111-at (PPP1R2) Source: Human protein phosphatase inhibitor 2 (PPP1R2) gene, exon 6 and complete cds.
U60521_at (CASP9) caspase 9, apoptosis-related cysteine 2s protease L19783_at (PIGH) phosphatidylinositol glycan, class H
X60487 at (H4/h) J04056~at (CBR1) carbonyl reductase 1 3o Sensitive Tumor (Low) and Resistant Tumor (High) U56998 at (CNK) cytokine-inducible kinase X68277-at (DUSP1) dual specificity phosphatase 1 U65092-at (MSG1) melanocyte specific gene 1 DOCSTOR: 639063\ 1 X01703 at Source: Human gene for alpha-tubulin (b alpha 1).
Table 2: genes that showed high expression in a sensitive tumor and low expression in a resistant tumor, or genes that s showed low expression in a sensitive tumor and high expression in a resistant tumor (genes that showed decreased expression in vitro by treatment with FR901228) Sensitive Tumor (High) and Resistant Tumor (Low) io X74987 s at (RNASELI) ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) inhibitor J03801 f at (LYZ) lysozyme (renal amyloidosis) U09578 at (MAPKAPK3) mitogen-activated protein kinase-activated protein kinase 3 15 D50678 at (LRP8) low density lipoprotein receptor- related protein 8 Sensitive Tumor (Low) and Resistant Tumor (High) Y10375 s at (SIRP-alpha 1) 20 214982 rnal at (MHC-encoded proteasome subunit gene LAMP7-El) alternative splicing X62048 at (WEE1) weel + (S. pombe) homolog X71874 cdsl at (PSMB10) proteasome (prosome, macropain) subunit, beta type, 10 2s U32849 at (NMI) N-myc (and STAT) interactor D55716 at (Plcdc47) Source: Human mRNA for Plcdc47, complete cds.
M98045 at (FPGS) folylpolyglutamate synthase U21551-at (ECA39) Source: Human ECA39 mRNA, complete cds.
3o U06681 at U07620 at (PRKM10) protein kinase mitogen-activated 10 (MAP
kinase) DOCSTOR: 639063\1 These genes suggest correlation with efficacy of FK228, and correlation with sensitivity or resistance to FK228, thus s indicating the possibility of these genes being utilizable as an efficacy predicting marker.
Industrial Applicability so The therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present invention, comprising, as an active ingredient, FK228 (particularly FR901228) or a salt thereof having a histone deacetylase inhibitory activity, have a superior antitumor is action not only in vitro but also in vivo. Therefore, they can be used clinically, particularly suitably for cancer treatment.
Using the evaluation method or screening method of the present invention, moreover, a histone deacetylase inhibitor capable of exerting an antitumor effect specific to a target tumor 2o cell can be found without actually administering the inhibitor to a human body.
Sequence Listing Free Text SEQ ID; No 1: oligonucleotide designed to act as a primer for 2s PCR of p21 mRNA.
SEQ ID; No 2: oligonucleotide designed to act as a primer for PCR of p21 mRNA.
SEQ ID; No 3: oligonucleotide designed to act as a primer for PCR of c-myc mRNA.
3o SEQ ID; No 4: oligonucleotide designed to act as a primer for PCR of c-myc mRNA.
SEQ ID; No 5: Xaa is an amino acid represented by the formula NHZC ( CHCH3 ) COOH .
DOCSTOR: 639063\1 The carboxyl group of the formula COOHCHZCH(CHCHC2H4SH)OH
is bonded with an amino group of Val, which is the first amino acid, a hydroxyl group is bonded with a carboxyl group of Val, which is the fourth amino acid, and an SH group is disulfide-s bonded with an SH group of Cys, which is the second amino acid.
This application is based on a patent application No.
2001-250846 filed in Japan, the contents of which are hereby incorporated by reference.
DOCSTOR: 639063\1
Example 1 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent (10% HCO-60/saline) was added. The mixture was sonicated to s allow for dissolution. A positive control substance Paclitaxel was dissolved in Cremophor EL/ethanol (1:1) solution to 24 mg/mL prior to the testing, and preserved in a refrigerator.
When in use, it was diluted with a 9-fold amount of physiological saline to 2.4 mg/mL (solvent component: 5%
to Cremophor EL-5% ethanol-90% saline).
(2) Test animal For antitumor test of the pharmaceutical agent, BALB/cANnNCrj-nu/nu mice (male, 6-week-old) were purchased from Charles River Japan and, after acclimation for not less Is than one week, used for the test. The mice were reared under an SPF environment and allowed a free access to water and feed.
(3) Test tumor Cultured human prostate cancer cell line (PC-3: available from Japanese Foundation for Cancer Research, Cancer Zo Chemotherapy Center, Fundamental Research) was subcutaneously implanted at 2-3x10' cells in a nude mouse. A grown solid tumor was subcultured not less than 3 generations and used for the test.
(4) Experimental implantation and grouping 2s A solid tumor subcultured in a nude mouse was subcutaneously implanted in the right back of a mouse as an about 3 mm square tumor tissue fragment. After the tumor implantation, when the tumor volume (1/2xlonger diameterxshorter diameter2) reached 100-300 mm3, the mice were 3o grouped into 6 mice per group to level the tumor size.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intravenously administered to an DOCSTOR: 639063\ l FR901228 administration group 3 times every 4 days (q4dx3) (3.2 and 1.8 mg/kg). Paclitaxel (24 mg/kg) was intravenously administered for 5 consecutive days (qdx5) to a positive control substance paclitaxel administration group. Only a s solvent (loo HCO-60/saline) was administered (q4dx3) to a control group. The amount of liquid for each administration was calculated (0.1 mL/10 g body weight) based on the body weight measured on the administration day. Note that 3.2 mg/kg/day (q4dx3) of FR901228 and 24 mg/kg/day (qdx5) of to Paclitaxel were the maximum tolerated doses (MTD) thereof.
(6) Measurement of tumor size and body weight The tumor size (longer diameter, shorter diameter) and body weight were measured twice a week from Day 0.
(7) Evaluation of antitumor effect is The level of tumor growth was evaluated based on the tumor growth rate (Relative Tumor Volume). The growth suppression rate was expressed in a relative proportion of tumor volume after day 0 to the tumor volume at Day 0 as 1.
The antitumor effect was determined on the 14th day (Day 14) 2o from the start of the administration of the pharmaceutical agent. When the proportion of the tumor growth rate of the pharmaceutical agent administration group to that of the control group (solvent administration), (T/C%), was not more than 50o and a significant difference (P<0.01) was found in zs the Mann Whitney U-test, the pharmaceutical agent was determined to be effective.
The results are shown in Fig. 1. FR901228 showed an antitumor effect in vivo against human prostate cancer.
Example 2 30 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent (10s HCO-60/saline) was added. The mixture was sonicated to allow for dissolution.
DOCSTOR: 639063\I
(2) Test animal For antitumor test of the pharmaceutical agent, Fox Chase C.B-17/Icr-SCID.Jcl mice (male, 6-week-old) were purchased from CLEA JAPAN INC. and, after acclimation for not less than s one week, used for the test. The mice were reared under an SPF
environment and allowed a free access to water and feed.
(3) Test tumor Cultured human lymphoma cell line (U937: obtained from Dr.
Minowada, Hayashibara Biochemical Laboratories, Inc.) was to cultured in RPMI (containing 10% FCS) and subcultured in vitro.
(4) Experimental implantation and grouping Cyclo phosphamide (Shionogi & Co., Ltd., 150 mg/kg) was intraperitoneally administered to mice. Lymphoma (1x107 cells) subcultured in vitro was intraperitoneally implanted the next is day. After the tumor implantation, the mice were grouped into 6 (control group 12) mice per group to level the body weight.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intraperitoneally administered to an 2o FR901228 administration group once or twice a week (0.1-1.0 mg/kg). Only a solvent (loo HCO-60/saline) was administered to a control group.
(6) Evaluation As an antitumor effect, the survival days of the mice 2s were counted.
The results are shown in Fig. 2. Fig. 2(a) shows the results of administration of FR901228 once a week, and Fig.
2(b) shows the results of administration of FR901228 twice a week. FR901228 showed an antitumor effect in vivo against 3o human lymphoma.
Example 3 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and a solvent DOCSTOR: 639063\1 (10% HCO-60/saline) was added. The mixture was sonicated to allow for dissolution. A positive control substance Paclitaxel was dissolved in Cremophor EL/ethanol (1:1) solution to 24 mg/mL prior to the testing, and preserved in a refrigerator.
s When in use, it was diluted with a 9-fold amount of physiological saline to 2.4 mg/mL (solvent component: 50 Cremophor EL-5o ethanol-90o saline).
(2) Test animal For antitumor test of the pharmaceutical agent, to BALB/cANnNCrj-nu/nu mice (male, 6-week-old) were purchased from Charles River Japan and, after acclimation for not less than one week, used for the test. The mice were reared under an SPF environment and allowed a free access to water and feed.
(3) Test tumor is Cultured human kidney cancer cell line 1 (ACHN: available from ATCC) and cultured human prostate cancer cell line 1 (PC-3: available from ATCC) were subcutaneously implanted at 2-3x10' cells in a nude mouse. A grown solid tumor was subcultured not less than 3 generations and used for the test.
2o (4) Experimental implantation and grouping A solid tumor subcultured in a nude mouse was subcutaneously implanted into the right back of a mouse as an about 3 mm square tumor tissue fragment. After the tumor implantation, when the tumor volume (1/2xlonger 2s diameterxshorter diameter2) reached 100-300 mm3, the mice were grouped into 6 mice per group to level the tumor size.
(5) Administration The administration was started on the day of grouping (Day 0). FR901228 was intravenously administered to an 3o FR901228 administration group 3 times every 4 days (q4dx3) (3.2 and 1.8 mg/kg). Paclitaxel was intravenously administered (24 mg/kg) for 5 consecutive days (qdx5) to a positive control substance Paclitaxel administration group. Only a solvent (10%
DOCSTOR: 639063\1 HCO-60/saline) was administered (q4dx3) to a control group.
The amount of liquid for each administration was calculated (0.1 mL/10 g body weight) based on the body weight measured on the administration day. Note that 3.2 mg/kg/day (q4dx3) of s FR901228 and 24 mg/kg/day (qdXS) of paclitaxel were MTDs thereof .
(6) Measurement of tumor size and body weight The tumor size (longer diameter, shorter diameter) and body weight were measured twice a week from Day 0.
to (7) Evaluation of antitumor effect The level of tumor growth was evaluated based on the tumor growth rate (Relative Tumor Volume). The growth suppression rate was expressed in a relative proportion of tumor volume after day 0 to the tumor volume at Day 0 as 1.
Is The antitumor effect was determined on the 14th day (Day 14) from the start of the administration of a pharmaceutical agent.
When the ratio of the pharmaceutical agent administration group to the tumor growth rate of the control group (solvent administration) (T/Co) was not more than 500, and a zo significant difference (P<0.01) was found in the Mann Whitney U-test, the pharmaceutical agent was determined to be effective.
The results are shown in Fig. 3. FR901228 showed a potent antitumor effect against PC-3 at the dose of 3.2 mg/kg 2s (Fig. 3(a)) but did not show an antitumor effect against ACHN
(Fig. 3(b)).
Example 4 (1) Preparation of pharmaceutical agent A necessary amount of FR901228 was weighed and dissolved 3o in a solvent (99.50 ethanol) to the concentration of 1 mg/mL.
Then, the solution was diluted with culture medium.
(2) Test tumor Cultured human cancer cell (PC-3 and ACHN) were cultured DOCSTOR: 639063\1 in DMEM (containing loo FCS).
(3) Culture and RNA extraction The cells were inoculated at 2x106 cells per a culture dish, and cultured in the presence of FR901228 (5 ng/mL) for a s given time. After culturing, RNA was extracted with a TRIZOL
reagent (GIBCO BRL) according to the operation manual.
(4) Real time PCR
RNA was subjected to reverse transcription using a Taq man reverse transcription reagent (PE Biosystem) according to io the operation manual. Thereafter, p21 gene was amplified using a SYBR green PCR master mix (PE Biosystem) and primer 5'-GGC
AGA CCA GCA TGA CAC ATT-3' (p21 upstream)(SEQ ID; No 1), 5'-GGA TTA GGG CTT CCT CTT GGA G-3' (SEQ ID; No 2) according to the operation manual, and detected with ABI 7700 PRISM
is sequence detector (PE Biosystem). The expression amount of p21 gene was calculated from a standard curve, divided by the expression amount of ~3-actin gene, which was used as an internal standard, and expressed in a standardized relative expression amount.
2o The results are shown in Fig. 4. By being contacted with FR901228 in vitro, PC-3 (Fig. 4(a)) showed an increased expression of the p21 gene with the lapse of time. In contrast, ACHN did not show increased expression of p21 gene (Fig. 4(b)).
When untreated, p21 gene showed little expression in PC-3 but 2s showed expression in ACHN (Fig. 4(c)).
Example 5 Human prostate.cancer PC-3 or kidney cancer ACHN was subcutaneously implanted in a nude mouse, and when the size of the tumor reached 100-300 mg, FR901228 (3.2 mg/kg) was 3o intravenously administered. The tumor was removed with the lapse of time, and after extracting RNA, the expression amount of p21 gene and c-myc gene was examined by real time PCR in the same manner as in Example 4. The c-myc gene was amplified DOCSTOR: 639063\1 using a SYBR green PCR master mix (PE Biosystem) and primer 5'-GAC AGA TCA GCA ACA ACC GAA A-3' (human c-myc upstream)(SEQ
ID; No 3), 5'-TTG TGT GTT CGC CTC TTG ACA T-3' (human c-myc downstream)(SEQ ID; No 4) according to the operation manual, s and detected with ABI 7700 PRISM sequence detector (PE
Biosystem).
The results are shown in Fig. 5. PC-3 showed increased expression of p21 gene in vivo with a peak at 3 hr after administration of FR901228 (Fig. 5(a)). In contrast, ACHN did 1o not show increased expression of p21 gene (Fig. 5(a)). While c-myc gene showed decreased expression in PC-3, it showed increased expression in ACHN (Fig. (b)).
Example 6: Analysis (in vitro) of gene expression by FK228 in tumor cell using a gene chip Is The effect of FK228 on in vitro gene expression in human tumor cell was analyzed using a gene chip.
<material ~ procedure>
(1) Test materials pharmaceutical agent FK228 (FR901228) 2o concentration at use: 50 ng/mL
preparation method: A 10 mg/mL solution was prepared with ethanol in advance and serially diluted with a culture medium to give 50 ng/mL solutions.
dosage form: solution (prepared when in use) 2s Cells used: human prostate cancer (PC-3), human lymphoma (U937), human kidney cancer (ACHN) Culture medium: DMEM (for PC-3, ACHN), RPMI1640 (for U937) Both obtained from Nikken Biomedical Laboratory, further containing FCS (Moregate) and Penicillin-Streptomycin 30 (ICN Biomedicals Inc.).
RNA extraction: RNeasy Mini Kit (50) (Qiagen) RNase, DNase free water (Life Technologies) DOCSTOR: 639063\ 1 DNA synthesis: Superscript Choice System (Life Technologies) Ethachinmate (Nippon gene) T7-(dT)24 Primer (Amersham Pharmacia) cRNA synthesis: BioArray RNA Transcript Labeling Kit (Amersham s Pharmacia) cRNA fragmentation: Trizma Base (SIGMA) glacial acetic acid (SIGMA) magnesium acetate (SIGMA) potassium acetate (SIGMA) to Hybridization: Eukaryotic Hybridization Control Kit (Amersham Pharmacia) 0.5M EDTA solution (SIGMA) MES Sodium Salt (SIGMA) MES Free Acid Monohydrate (SIGMA) is Herring Sperm DNA (Promega) Acetylated Bovine Serum Albumin Soln. (Life Technologies) Staining: Phycoerythrin-Streptavidin (Molecular Probes) Goat IgG, Reagent Grade (SIGMA) 2o Anti-streptavidin ab (goat), biotinylated (Vector Lab) Chip used: HuGeneFL array (Amersham Pharmacia) (2) Cell preparation and RNA extraction Human tumor cells (PC-3, U937, ACHN) that reached zs confluent in F75 flasks were subjected to a trypsin treatment to give single cell suspensions, which were inoculated to five F75 flasks and cultured for 24 hr. The culture medium was discarded and a fresh culture medium (18 mL) and a 10-fold concentration (50 ng/mL) of an FR901228 solution (2 mL) were 3o added. The mixture was cultured in a COZ incubator at 37°C for a given time (0, 1, 3, 12 and 24 hr). After the completion of the culturing, the culture medium was discarded and total RNA
was extracted according to the protocol of RNeasy Mini Kit DOCSTOR: 639063\ 1 (50) (Qiagen). RNA was quantified and confirmed by electrophoresis.
(3) Synthesis of cRNA
According to the GeneChip manual, Chapter 2 - Chapter 4, s and the manuals of RNeasy Mini Kit and RNA Transcript Labeling Kit, RNA was purified, cDNA was synthesized, cRNA was synthesized and cRNA was fragmented.
(4) Hybridization, Washing-staining, Scanning Hybridization, washing-staining and scanning were io conducted according to the GeneChip manual Chapter 5 - Chapter 7.
(5) Analysis Analyzed using GeneSpring (microarray data analysis soft:
manufactured by Silicon Genetics).
is <Results>
Human prostate cancer PC-3 and human lymphoma U937, which are FK228 sensitive tumor cells, and human kidney cancer ACHN, which is an FK228 resistant tumor cell, were brought into contact in vitro with FK228 with the lapse of time, and RNA
2o was extracted thereafter, and 7070 genes detectable using GeneChip were examined for genes showing change in the expression due to FK228. Such genes were analyzed according to the following procedures.
Analysis 1: Selection of gene that shows change in expression 2s due to inhibition of histone deacetylase.
For limiting the gene that shows change in expression due to FK228, a gene showing linear change of expression was selected (analysis condition of GeneSpring: gene that shows change in expression by 0.5 time or more or 0.5 time or less so at any time).
Analysis 2: Selection of gene involved in efficacy When contacted with FK228 for 72 hr, growth suppression effect on PC-3, U937 and ACHN in ICSO value was 3.17, 3.20 and DOCSTOR: 639063\1 4.25 ng/mL, respectively, showing almost the same degree of growth suppressive effect on these tumor cells. From these results, the genes relating to the growth suppression were considered to commonly show change in expression in any cell.
s There were found 105 genes that commonly showed increased expression in these three kinds of human tumor cells and 100 genes that commonly showed decreased expression in these three kinds of human tumor cells.
Example 7: Analysis of gene expression (in vivo) by FK228 in Io tumor cell using a gene chip The effect of FK228 on in vivo gene expression in human tumor cell was analyzed using a gene chip.
<material ~ procedure>
(1) Test materials i5 Pharmaceutical agent FK228 (FR901228) dose: 10 mg/kg administration dose: 10 mL/kg solvent: 10% HCO-60/saline solution dosage form: solution (prepared when in use) 2o Tumor cell: human prostate cancer PC-3 (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) human gastric cancer SC-6; obtained from Central Institute for Experimental Animals (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) 2s human kidney cancer ACHN (tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) human kidney cancer A498; obtained from ATCC
(tumor fragment 3 mmx3 mmx3 mm/mouse implantation site s.c.) 3o Subcultured animal: male BALB c/nu/nu RNA extraction: RNeasy Mini Kit (50) (Qiagen) RNase, DNase free water (Life Technologies) DOCSTOR: 63906311 DNA synthesis: Superscript Choice System (Life Technologies) Ethachinmate (Nippon gene) T7-(dT)24 Primer (Amersham Pharmacia) cRNA synthesis: BioArray RNA Transcript Labeling Kit (Amersham s Pharmacia) cRNA fragmentation: Trizma Base (SIGMA) glacial acetic acid (SIGMA) magnesium acetate (SIGMA) potassium acetate (SIGMA) to Hybridization: Eukaryotic Hybridization Control Kit (Amersham Pharmacia) 0.5 M EDTA solution (SIGMA) MES Sodium Salt (SIGMA) MES Free Acid Monohydrate (SIGMA) is Herring Sperm DNA (Promega) Acetylated Bovine Serum Albumin Soln. (Life Technologies) Chip used: HuGeneFL array (Amersham Pharmacia) (2) Cell preparation and RNA extraction 20 A 3 mm square tumor fragment (PC-3, SC-6, ACHN, A498) was subcutaneously implanted in a nude mouse and, when the tumor reached about 100 mg (longer diameter 9 mm, shorter diameter 8 mm), FR901228 (10 mg/kg) was intravenously administered. At 0, 0.5, 1, 2 and 4 hr after administration of FR901228, the tumor as was removed and total RNA was extracted according to the protocol of RNeasy Mini Kit (50) (Qiagen). RNA was quantified and confirmed by electrophoresis.
(3) Synthesis of cRNA
According to the GeneChip manual, Chapter 2 - Chapter 4, 3o and the manuals of RNeasy Mini Kit and RNA Transcript Labeling Kit, RNA was purified, cDNA was synthesized, cRNA was synthesized and cRNA was fragmented.
(4) Hybridization, Washing-staining, Scanning DOCSTOR: 639063\1 Hybridization, washing-staining and scanning were conducted according to the GeneChip manual Chapter 5 - Chapter 7.
(5) Analysis s Analyzed using GeneSpring (microarray data analysis soft:
manufactured by Silicon Genetics).
<Results>
FR901228 (10 mg/kg) was intravenously administered to human prostate cancer PC-3, human gastric cancer SC-6, human to kidney cancer ACHN and human kidney cancer A498-carrying cancer mice and tumor was removed with the lapse of time (0, 0.5, 1, 2 and 4 hr). RNA was then extracted and 7070 genes detectable using GeneChip were examined for genes showing change in the expression due to FR901228.
Is The growth suppression rate of human prostate cancer PC-3, human gastric cancer SC-6, human kidney cancer ACHN and human kidney cancer A498 by the administration of FR901228 (3.2 mg/kg) was 98%, 840, 20o and 290, respectively. Therefore, PC-3 and SC-6 were determined to be FK228 sensitive tumors, and 2o ACHN and A498 were determined to be FK228 resistant tumors.
Example 8: Mode of gene expression in FK228 sensitive tumor and FK228 resistant tumor The correlation between the mode of gene expression and efficacy of FK228 was examined in the tumor confirmed to be 2s sensitive or resistant in Example 7. Close attention was given to the genes (105 genes) that showed increased expression by FK228 treatment and genes (100 genes) that showed decreased expression thereby as demonstrated in the in vitro test in Example 6, and the correlation between these genes and 3o efficacy was studied. Furthermore, a gene that showed high expression in a sensitive tumor and low expression in a resistant tumor, and a gene that showed low expression in a sensitive tumor and high expression in a resistant tumor were DOCSTOR: 6390630 searched for. As a result, of the 105 genes that showed increased expression in vitro by treatment with FR901228, 6 genes were found to show high expression in a sensitive tumor and low expression in a resistant tumor, and 4 genes were s found to show low expression in a sensitive tumor and high expression in a resistant tumor (Table 1). In addition, of the 100 genes that showed decreased expression in vitro by treatment with FR901228, 4 genes were found to show high expression in a sensitive tumor and low expression in a to resistant tumor, and 9 genes were found to show low expression in a sensitive tumor and high expression in a resistant tumor (Table 2).
Table 1: genes that showed high expression in a sensitive is tumor and low expression in a resistant tumor, or genes that showed low expression in a sensitive tumor and high expression in a resistant tumor (genes that showed increased expression in vitro by treatment with FR901228) 2o Sensitive Tumor (High) and Resistant Tumor (Low) M13686,s_at (SFTP1) pulmonary surfactant-associated protein U68111-at (PPP1R2) Source: Human protein phosphatase inhibitor 2 (PPP1R2) gene, exon 6 and complete cds.
U60521_at (CASP9) caspase 9, apoptosis-related cysteine 2s protease L19783_at (PIGH) phosphatidylinositol glycan, class H
X60487 at (H4/h) J04056~at (CBR1) carbonyl reductase 1 3o Sensitive Tumor (Low) and Resistant Tumor (High) U56998 at (CNK) cytokine-inducible kinase X68277-at (DUSP1) dual specificity phosphatase 1 U65092-at (MSG1) melanocyte specific gene 1 DOCSTOR: 639063\ 1 X01703 at Source: Human gene for alpha-tubulin (b alpha 1).
Table 2: genes that showed high expression in a sensitive tumor and low expression in a resistant tumor, or genes that s showed low expression in a sensitive tumor and high expression in a resistant tumor (genes that showed decreased expression in vitro by treatment with FR901228) Sensitive Tumor (High) and Resistant Tumor (Low) io X74987 s at (RNASELI) ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) inhibitor J03801 f at (LYZ) lysozyme (renal amyloidosis) U09578 at (MAPKAPK3) mitogen-activated protein kinase-activated protein kinase 3 15 D50678 at (LRP8) low density lipoprotein receptor- related protein 8 Sensitive Tumor (Low) and Resistant Tumor (High) Y10375 s at (SIRP-alpha 1) 20 214982 rnal at (MHC-encoded proteasome subunit gene LAMP7-El) alternative splicing X62048 at (WEE1) weel + (S. pombe) homolog X71874 cdsl at (PSMB10) proteasome (prosome, macropain) subunit, beta type, 10 2s U32849 at (NMI) N-myc (and STAT) interactor D55716 at (Plcdc47) Source: Human mRNA for Plcdc47, complete cds.
M98045 at (FPGS) folylpolyglutamate synthase U21551-at (ECA39) Source: Human ECA39 mRNA, complete cds.
3o U06681 at U07620 at (PRKM10) protein kinase mitogen-activated 10 (MAP
kinase) DOCSTOR: 639063\1 These genes suggest correlation with efficacy of FK228, and correlation with sensitivity or resistance to FK228, thus s indicating the possibility of these genes being utilizable as an efficacy predicting marker.
Industrial Applicability so The therapeutic agent for prostate cancer and the therapeutic agent for malignant lymphoma of the present invention, comprising, as an active ingredient, FK228 (particularly FR901228) or a salt thereof having a histone deacetylase inhibitory activity, have a superior antitumor is action not only in vitro but also in vivo. Therefore, they can be used clinically, particularly suitably for cancer treatment.
Using the evaluation method or screening method of the present invention, moreover, a histone deacetylase inhibitor capable of exerting an antitumor effect specific to a target tumor 2o cell can be found without actually administering the inhibitor to a human body.
Sequence Listing Free Text SEQ ID; No 1: oligonucleotide designed to act as a primer for 2s PCR of p21 mRNA.
SEQ ID; No 2: oligonucleotide designed to act as a primer for PCR of p21 mRNA.
SEQ ID; No 3: oligonucleotide designed to act as a primer for PCR of c-myc mRNA.
3o SEQ ID; No 4: oligonucleotide designed to act as a primer for PCR of c-myc mRNA.
SEQ ID; No 5: Xaa is an amino acid represented by the formula NHZC ( CHCH3 ) COOH .
DOCSTOR: 639063\1 The carboxyl group of the formula COOHCHZCH(CHCHC2H4SH)OH
is bonded with an amino group of Val, which is the first amino acid, a hydroxyl group is bonded with a carboxyl group of Val, which is the fourth amino acid, and an SH group is disulfide-s bonded with an SH group of Cys, which is the second amino acid.
This application is based on a patent application No.
2001-250846 filed in Japan, the contents of which are hereby incorporated by reference.
DOCSTOR: 639063\1
Claims (34)
1. An agent for treating prostate cancer, which comprises a compound represented by the formula (I) or a salt thereof as an active ingredient.
2. The agent of claim 1, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
3. The agent of claim 1, which has an antitumor action in vivo.
4. An agent for treating malignant lymphoma other than T cell lymphoma, which comprises a compound represented by the formula (I) or a salt thereof as an active ingredient.
5. The agent of claim 4, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
6. The agent of claim 4, which has an antitumor action in vivo.
7. A pharmaceutical composition for treating prostate cancer, which comprises a compound represented by the formula (I) or a salt thereof, and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
9. The pharmaceutical composition of claim 8, which has an antitumor action in vivo.
10. A pharmaceutical composition for treating malignant lymphoma other than T cell lymphoma, which comprises a compound represented by the formula (I) or a salt thereof, and a pharmaceutically acceptable carrier.
11. The pharmaceutical composition of claim 10, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
12. The pharmaceutical composition of claim 10, which has an antitumor action in vivo.
13. A method for treating prostate cancer, which comprises administering an effective amount of a compound represented by the formula (I) or a salt thereof to a patient.
14. The method of claim 13, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
15. A method for treating malignant lymphoma other than T cell lymphoma, which comprises administering an effective amount of a compound represented by the formula (I) or a salt thereof to a patient.
16. The method of claim 15, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
17. Use of a compound represented by the formula (I) or a salt thereof for the production of an agent for treating prostate cancer.
18. The use of claim 17, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
19. The use of claim 17, wherein the agent for treating prostate cancer has an antitumor action in vivo.
20. Use of a compound represented by the formula (I) or a salt thereof for the production of an agent for treating malignant lymphoma other than T cell lymphoma.
21. The use of claim 20, wherein the compound represented by the formula (I) is a compound represented by the formula (II)
22. The use of claim 20, wherein the agent for treating malignant lymphoma other than T cell lymphoma has an antitumor action in vivo.
23. A commercial package comprising the pharmaceutical composition of claim 7 and a written matter stating that the pharmaceutical composition can or should be used for treating prostate cancer.
24. A commercial package comprising the pharmaceutical composition of claim 10 and a written matter stating that the pharmaceutical composition can or should be used for treating malignant lymphoma other than T cell lymphoma.
25. A method for evaluating an antitumor effect of a histone deacetylase inhibitor, which comprises at least a step of treating a test cell with a histone deacetylase inhibitor, and a step of measuring change in the expression of a specific gene in the test cell before and after the treatment with said inhibitor, and comparing the both expression amounts.
26. The method of claim 25, wherein the specific gene is a p21 gene and/or c-myc gene.
27. The method of claim 25 or 26, wherein the histone deacetylase inhibitor is a compound represented by the formula (I) or a salt thereof.
28. The method of claim 27, wherein the histone deacetylase inhibitor is a compound represented by the formula (IT) or a salt thereof.
29. A method for evaluating an antitumor effect of a histone deacetylase inhibitor, which comprises at least a step of treating a test cell with a histone deacetylase inhibitor, and a step of measuring change in the expression of a specific protein in the test cell before and after the treatment with said inhibitor, and comparing the both expression amounts.
30. The method of claim 29, wherein the specific protein is p21 and/or c-myc.
31. The method of claim 29 or 30, wherein the histone deacetylase inhibitor is a compound represented by the formula (I) or a salt thereof.
32. The method of claim 31, wherein the histone deacetylase inhibitor is a compound represented by the formula (II) or a salt thereof.
33. A method for screening a histone deacetylase inhibitor having a site-specific antitumor activity, which comprises use of the method of any of claims 25 to 32.
34. A method for obtaining a gene capable of being an index for predicting the efficacy of FK228, which comprises at least (1) a step of treating an FK228 sensitive tumor cell and an FK228 resistant tumor cell with FK228, (2) a step of selecting genes that show increased or decreased expression in step (1) above, and (3) a step of selecting, from the genes selected in step (2) above, (i) a gene that shows increased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, (ii) a gene that shows increased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell, (iii) a gene that shows decreased expression due to the treatment with FK228, higher expression in the FK228 sensitive tumor cell and lower expression in the FK228 resistant tumor cell, or (iv) a gene that shows decreased expression due to the treatment with FK228, lower expression in the FK228 sensitive tumor cell and higher expression in the FK228 resistant tumor cell.
Applications Claiming Priority (3)
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|---|---|---|---|
| JP2001250846 | 2001-08-21 | ||
| JP2001-250846 | 2001-08-21 | ||
| PCT/JP2002/008355 WO2003015810A1 (en) | 2001-08-21 | 2002-08-20 | Medicinal use of histone deacetylase inhibitor and method of evaluating antitumor effect thereof |
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| PT (1) | PT1426054E (en) |
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-
2002
- 2002-08-20 JP JP2003520768A patent/JP4238728B2/en not_active Expired - Lifetime
- 2002-08-20 PL PL368708A patent/PL210794B1/en unknown
- 2002-08-20 PT PT02760663T patent/PT1426054E/en unknown
- 2002-08-20 WO PCT/JP2002/008355 patent/WO2003015810A1/en active Application Filing
- 2002-08-20 AT AT02760663T patent/ATE526029T1/en active
- 2002-08-20 RU RU2004108137/15A patent/RU2298414C2/en not_active IP Right Cessation
- 2002-08-20 US US10/486,833 patent/US8470783B2/en active Active
- 2002-08-20 ES ES02760663T patent/ES2371630T3/en not_active Expired - Lifetime
- 2002-08-20 EP EP02760663A patent/EP1426054B1/en not_active Expired - Lifetime
- 2002-08-20 DK DK02760663.1T patent/DK1426054T3/en active
- 2002-08-20 CA CA002457043A patent/CA2457043A1/en not_active Abandoned
- 2002-08-20 MX MXPA04001612A patent/MXPA04001612A/en active IP Right Grant
- 2002-08-20 HU HU0400668A patent/HU229521B1/en not_active IP Right Cessation
-
2004
- 2004-02-20 NO NO20040731A patent/NO331682B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| US8470783B2 (en) | 2013-06-25 |
| EP1426054B1 (en) | 2011-09-28 |
| EP1426054A1 (en) | 2004-06-09 |
| JP4238728B2 (en) | 2009-03-18 |
| JPWO2003015810A1 (en) | 2004-12-02 |
| ATE526029T1 (en) | 2011-10-15 |
| RU2004108137A (en) | 2005-05-10 |
| US20050191713A1 (en) | 2005-09-01 |
| HU229521B1 (en) | 2014-01-28 |
| PT1426054E (en) | 2011-12-06 |
| RU2298414C2 (en) | 2007-05-10 |
| PL368708A1 (en) | 2005-04-04 |
| HUP0400668A2 (en) | 2005-01-28 |
| NO331682B1 (en) | 2012-02-20 |
| NO20040731L (en) | 2004-04-20 |
| ES2371630T3 (en) | 2012-01-05 |
| EP1426054A4 (en) | 2006-09-13 |
| MXPA04001612A (en) | 2004-07-08 |
| PL210794B1 (en) | 2012-03-30 |
| HUP0400668A3 (en) | 2012-09-28 |
| DK1426054T3 (en) | 2011-12-12 |
| WO2003015810A1 (en) | 2003-02-27 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |
Effective date: 20140422 |